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https://f1000research.com/articles/10-373/v1	11 May 21	{ "type": "Brief Report", "title": "Multi-modality detection of SARS-CoV-2 in faecal donor samples for transplantation and in asymptomatic emergency surgical admissions", "authors": [ "Susan E. Manzoor", "Shafquat Zaman", "Celina Whalley", "David Inglis", "Andrew Bosworth", "Michael Kidd", "Sahida Shabir", "Nabil Quraishi", "Christopher A. Green", "Tariq Iqbal", "Andrew D. Beggs", "Susan E. Manzoor", "Shafquat Zaman", "Celina Whalley", "David Inglis", "Andrew Bosworth", "Michael Kidd", "Sahida Shabir", "Nabil Quraishi", "Christopher A. Green", "Tariq Iqbal" ], "abstract": "Background: Faecal transplantation is an evidence-based treatment for Clostridioides difficile. Patients infected with SARS-CoV-2 have been shown to shed the virus in stool for up to 33 days, well beyond the average clearance time for upper respiratory tract shedding. We carried out an analytical and clinical validation of reverse-transcriptase quantitative (RT-qPCR) as well as LAMP, LamPORE and droplet digital PCR in the detection of SARS-CoV-2 RNA in stool from donated samples for faecal microbiota transplantation (FMT), spiked samples and asymptomatic inpatients in an acute surgical unit. Methods: Killed SARS-CoV-2 viral lysate and extracted RNA was spiked into donor stool & FMT and a linear dilution series from 10-1 to 10-5 and tested via RT-qPCR, LAMP, LamPORE and ddPCR against SARS-CoV-2. Patients admitted to the critical care unit with symptomatic SARS-CoV-2 and sequential asymptomatic patients from acute presentation to an acute surgical unit were also tested. Results: In a linear dilution series, detection of the lowest dilution series was found to be 8 copies per microlitre of sample. Spiked lysate samples down to 10-2 dilution were detected in FMT samples using RTQPCR, LamPORE and ddPCR and down to 10-1 with LAMP. In symptomatic patients 5/12 had detectable SARS-CoV-2 in stool via RT-qPCR and 6/12 via LamPORE, and in 1/97 asymptomatic patients via RT-qPCR. Conclusion: RT-qPCR can be detected in FMT donor samples using RT-qPCR, LamPORE and ddPCR to low levels using validated pathways. As previously demonstrated, nearly half of symptomatic and less than one percent of asymptomatic patients had detectable SARS-CoV-2 in stool.", "keywords": [ "Faecal microbiota transplant", "SARS-CoV-2", "Detection" ], "content": "Introduction\n\nSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, which causes coronavirus disease 2019 (COVID-19), first emerged in Wuhan, China in late 20191 and is responsible for nearly 1.5 million deaths worldwide. SARS-CoV-2 is primarily transmitted via respiratory droplets and direct contact routes between asymptomatic and symptomatic individuals. Although faeco-oral transmission has not been documented with this virus2, it is one of the main forms of transmission with other similar single-stranded RNA viruses, such as norovirus3. Multiple lines of evidence suggest that the gastrointestinal tract may form a reservoir for SARS-CoV-2 with the potential for infection and transmission4. The virus enters the host by binding its S1 “spike” glycoprotein to angiotensin-converting enzyme 2 (ACE2) in epithelial tissues which is avidly expressed in the ileum5. Furthermore, gastro-intestinal symptoms such as diarrhoea are not uncommon in patients infected with SARS-CoV-2 affecting up to 40% patients admitted to hospital. Several studies have identified that over 40% of patients with detectable SARS-CoV-2 RNA from nasopharyngeal swabs, will also have detectable viral RNA in faecal samples6. Moreover, faecal shedding of viral RNA has been shown to persist for up to 33 days after clearance from respiratory samples6. It is not known for certain whether SARS-CoV-2 present in faeces represents live and transmissible virus7, although early evidence suggests that this is possible in some and there remains uncertainty regarding the role of the gut in COVID-19 pathogenesis, potential for faeco-oral transmission of the virus and future outbreaks of infection in institutions such as hospitals and care homes.\n\nFaecal microbiota transplantation (FMT) involves the transplantation of processed faecal samples8 from healthy donors to individuals with disease associated with imbalance in the gut microbiome. In recent years FMT has transformed the treatment of patients with Clostridioides difficile infection (CDI)9, especially in those with recurrent or refractory disease. Although FMT donors and their stool donations undergo screening to good manufacturing procedure (GMP) standards for pathogens of potential significance, such as multi-drug resistant Enterobacteriaceae, some risks of this remain10. During the current pandemic many stool banks have need to stop providing FMT due to the potential risk of transmission of SARS-CoV-2 from asymptomatic donors and, earlier this year the Federal Drug Administration mandated that only stool donated prior to 1st December 2019 could be used for FMT. A recent international consensus paper recommenced direct stool testing for the presence of SARS-CoV-2 would be needed for safe FMT supply in the COVID-19 era 202011,12.\n\nIn this paper we present the first the results of testing FMT donor stool for the presence of SARS-CoV-2 virus using an internally developed assay based on an existing CE-IVD marked product as well as various novel diagnostics that have been developed in response to the pandemic. We also report using this to test asymptomatic acute patients admitted to hospital with COVID-19 as part of the ‘second spike’ of the pandemic in 2020 in order to understand the prevalence of faecal SARS-CoV-2 detection in this cohort.\n\n\nMethods\n\nStool samples were collected with full written consent under an existing gut microbial profiling study with ethical approval from Yorkshire & The Humber - Bradford Leeds Research Ethics Committee (16/YH/0100). Samples were obtained from 12 symptomatic COVID-19 patients admitted to hospital who tested positive for SARS-CoV-2 RNA with PCR testing on naso-pharyngeal swabs and a further 97 asymptomatic patients presenting to the Surgical Assessment Unit of Sandwell and West Birmingham NHS Trust from September-November 2020 (a time of increasing prevalence).\n\nSamples used for spiking experiments were taken from pre-existing stocks at the University of Birmingham Microbiome Treatment Centre FMT bank. These were from a batch collected and stored in 2017, prior to the emergence of the SARS-CoV-2 virus. These samples were collected in readiness for use in a trial of FMT in inflammatory bowel disease (STOP-COLITIS 2015-005753-12). For this study, FMT was manufactured over a 10-day donation period and retention and stool study samples were collected and stored in accordance with an MHRA approved GMP process REC and ethical approval (17/EM/0274).\n\nQuantitative PCR was used to analyse both COVID-19 patients and pre-COVID FMT following “spiking”, with varying concentrations of SARS-CoV-2 inactivated lysate (cell culture in Qiagen AL buffer). Purified RNA extracted from an aliquot of the lysate was also analysed using quantitative PCR as an additional positive control to determine whether RNA could be recovered and detected, and to determine the limit of the assay. A negative control of nuclease-free water was incorporated into the assay.\n\nLive SARS-CoV-2 England/2/2020(VE6-T) virus was isolated from infected VE6-T cells, then inactivated in Qiagen lysis buffer AL and frozen at -80oC. Viral RNA was purified from 500µL of cell lysate using a QIAmp Qiagen kit, then diluted serially to 10-5 in nuclease free water. Viral cell lysate was also diluted to 10-5 serially ten-fold in nuclease free water.\n\nThis dilution series of both lysate and RNA were then used to spike aliquots of faecal stool and FMT samples in duplicate. Extraction control samples consisting of 10-fold dilutions of cell lysate and RNA were also prepared.\n\nRNA was extracted from 0.2-0.25g of stool. All stool and FMT ‘spiked’ samples, along with the extraction control samples were lysed using bead beating PM1 buffer (containing guanidinium thiocyanate, Qiagen) to inactivate the virus prior to extraction under containment level 2+ (CL2+) conditions. A RNeasy PowerMicrobiome kit (Qiagen, Hilden) was then used for viral RNA extraction and purification of total RNA from stool with on-column DNase treatment. A subset of samples underwent quality assessment using an Agilent Tapestation 2200 with RNA Broad Range (BR) detection kit in order to understand the effects of RNA extraction upon stool samples.\n\nTwo real-time PCR detection kits for the detection of SARS-CoV-2 were used for the detection of virus in FMT and faecal samples and the extraction control samples: VIASURE SARS-CoV-2 Real Time PCR Detection kit (CerTest Biotec S.L.)13 and the WHO E Gene Assay Test14, in accordance with manufacturer instructions.\n\nViral RNA extracted from the spiked samples and extraction control samples underwent reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) using primers against the orf1ab polyprotein, nucleoplasmid structural protein (N) and the envelope membrane protein (E), which have been previously validated for the detection of SARS-CoV-215. Samples underwent analysis on a ThermoFisher QuantStudio 5 instrument. The thermocycling conditions consisted of an initial reverse transcription step at 45oC for 15 minutes, an initial denaturation step at 95oC for 2 minutes, then 45 cycles of 95oC for 10 seconds followed by 60oC for 60 seconds.\n\nThe level of fluorescence produced was analysed using ThermoFisher Connect (TM) software, and a sample was considered positive if the Ct value obtained was less than 38 in any of the gene targets (ORF1ab, N or E). Positive and negative controls were used in all reactions as well as an RNA extraction control, consisting of spiked viral lysate in buffer AVL.\n\nDroplet digital real-time PCR assays using target RNA, primers (450nM), fluorescent-labelled probes (200nM) and Bio-Rad ddPCR supermix were prepared. For use in this protocol, the E gene primers and 6-FAM labelled probe (used in the WHO E gene assay test14) were used with the One Step RT-ddPCR Advanced Kit (BioRad Laboratories). Samples were fractioned into 20,000 nanolitre sized water-in-oil droplets using QX200 Droplet generator (BioRad Laboratories). The droplets were transferred in a 96-well plate to a thermal cycler where PCR amplification of the template occurs within each droplet. Following PCR each droplet was analysed individually on the QX200 Droplet Reader (BioRad Laboratories) and the fraction of PCR-positive and PCR-negative droplets in the original sample were counted using QuantaSoft software. The data were analysed using Poisson distribution statistics to determine the concentration of target DNA in the original sample in absolute copies/ml.\n\nThe LamPORE SARS-CoV-2 assay utilises a combination of reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) and Nanopore sequencing technology (Oxford Nanopore Technologies, Oxford), as described previously16,17. For the SARS-CoV-2 the target regions N2, E1 and ORF1a genes each span approximately 180bp of the viral genome. Briefly, the viral RNA genome was reverse transcribed into cDNA, which was then amplified using strand-displacement polymerase to generate concatenated copies of the original target region. The DNA sequences produced (a 2kb concatamer with sequence for 180bp target region) were then aligned against the SARS-CoV-2 genome using a custom algorithm provided with the LamPORE system.\n\nIn order to understand the utility of a rapid assay to detect SARS-CoV-2 in stool, extracted faecal RNA underwent testing using the Optigene RNA-LAMP kit targeted against ORF1ab, under the manufacturer’s instructions for use.\n\n\nResults\n\nTo evaluate the analytical sensitivity of the assay and determine the analytical limit of detection, virus derived from in vitro cell culture was prepared in two different ways. Lysate was created, by adding equal volumes of cell culture supernatant containing live virus to a Buffer AL (Qiagen), a lysis buffer preparation containing guanidinium thiocyanate designed to both inactivate the virus rendering it safe to handle, and to stabilise RNA to protect it from RNAse mediated degradation.\n\nIt is recognised that re-extraction of purified RNA reduces the yield of RNA recovered, and unprotected RNA spiked into a matrix such as stool or FMT preparation is likely to degrade rapidly. To prevent this, lysate preparation was also spiked into the stool and FMT preparations. The lysate is less vulnerable as viral RNA is associated with proteins that serve to shield the virus and the addition of guanidinium thiocyanate even at low quantities serves to reduce the activity of RNAse degradative enzymes.\n\nFurthermore, the preparation method for extracting the RNA from the stool and FMT specimens requires mechanical homogenisation, which serves to further fragment and degrade RNA, even in samples such as that introduced in the lysate. When fragment analysis was carried out, we found RNA integrity number (RIN) of <2 in all samples, suggesting RNA processed in this way is highly degraded.\n\nAs anticipated, spiked RNA into faecal samples yielded poor results (Table 2 and Table 3), while the lysate was detectable at 3/5 dilutions. RNA extracted from the FMT and stool where lysate was used as the ‘spike in’, and was then tested using RT-ddPCR to determine the copy number recovered in this experiment. The lowest copy number recoverable from this dilution series was 8 copies of virus genome per reaction (or 0.4 copies per microliter of recovered RNA), equating to a limit of detection for the workflow (extraction and ViaSure RT-qPCR) of 8 copies per microliter of recovered RNA. The approximate limit of detection based on this data can be expressed as copies per gram of faecal preparation and is 204 copies of virus in 1g of faecal preparation using the following calculation:\n\n8 copies x 5 = 40 copies per 100ul (total eluted volume)\n\n* 1.3(estimated yield reduction from extraction)\n\n= 52 copies per elution. 52 copies = 0.25g input (extract weight) x 4\n\n= 204 copies of virus per 1g of faecal preparation.\n\nUsing the Viasure Real time PCR detection kit, amplification of the SARS-CoV-2 orf1ab and N genes were detected in the 10-1 and 10-2 lysate spiked faecal and FMT samples as well as the lysate extraction controls at all dilutions. Amplification in 10-3 to 10-5 concentrations for the lysate spiked samples was undetected. Similarly, using the WHO E gene Real-Time PCR detection assay, amplification of Envelope gene (E gene) of SARS-CoV-2.was detected in 10-1 and 10-2 lysate spike faecal samples and10-1 and 10-3 lysate spike FMT samples. Using the LamPORE SARS-CoV-2 protocol, amplification orf1ab, E and N genes were detected in 10-1 and 10-2 lysate spike faecal samples and10-1 and 10-3 lysate spike FMT samples. No signal was seen in any negative controls (Table 3).\n\nThe results of the digital droplet RT-qPCR correlated negatively with N gene results from the ViaSure assay (Pearson’s moment correlation coefficient r= -0.89) indicating good correlation of Ct values with viral content as assessed by the dd-rt-PCR experiment. LAMP only detected 10-1 spiked lysate concentration within the stool samples, with no virus detected in any other samples, likely due to template degradation during RNA extraction.\n\nOf the 12 symptomatic COVID-19 patients who provided stool samples (Table 1 and Table 4), 41.6% (5/12) had SARS CoV-2 RNA detectable in their stool via RT-QPCR , 5/12 (41.6%) via LamPORE and 6/12 (50%) via droplet digital qPCR. Of these patients 25% (3/12) reported gastrointestinal (GI) symptoms and of note, none of the patients with RNA shedding in their stool reported GI symptoms. The average age of the 12 patients was 55 and the median age was also 55. The majority of the patients 92% (12/13) recovered from their illness and were discharged, with one patient dying from their infection.\n\nUD: Undetermined/Undetected- Below lower detection limits repeated qPCR on this samples gave UD results.\n\nUD: Undetermined- Below lower detection limits\n\nInconclusive amplification\n\nUD: Undetermined- Below lower detection limits\n\n• E gene positive control 10-3 ATCC control\n\nWe collected and analysed stool samples from a total of 97 ‘asymptomatic’ patients presenting to SWBH over a 3-month period. This included a mix of new presentations to the surgical admissions unit (SAU) under various surgical specialities and existing inpatients. The mean age of our cohort was 65 years (range 20 – 94), median of 68 years. This included 55 (57%) females and 42 (43%) males. The majority of our participants were White-British – accounting for 74% of the total, with smaller numbers of Asian-British and Black-British patients. The majority of our patients (74%) with various co-morbidities presented with abdominal pain and were admitted under general surgery. Smaller numbers were under the care of urology, trauma and orthopaedics and medicine. Approximately 22% (21) of our patients had a stoma (ileostomy or colostomy).\n\nEighty (80%) percent (78) of our cohort denied symptoms of COVID-19 in the two weeks leading to hospital attendance/admission. Of the remainder, 18 (19%) complained of suffering with diarrhoea in the last 14 days. None of our patients had a fever, new onset cough, anosmia or ageusia.\n\nThe majority of our patients had a nasopharyngeal swab taken on admission to the hospital as per local Trust protocol. 78 (80%) of these returned a negative swab result, 11 (11%) were rejected by the laboratory (inadequate labelling), and in 5 the swab was either not collected or declined by the patient. Only 1 patient had a positive swab result. Using QPCR, 0/96 nasopharyngeal swab negative patients had detectable SARS-CoV-2 virus in their stool samples. In one patient who was swab positive 25 days previously (27 days post symptom onset) there was detectable SARS CoV-2 RNA in his stool with Ct value of 32.37 (ORF1ab gene) and 23.23 (N gene).\n\n\nConclusions\n\nWe have demonstrated with a CE-IVD marker two gene RT-qPCR assay, amplification was detected for both the ORF1ab and N genes in both faecal and FMT lysate spike replicates at 10-1 and 10-2 dilutions with lower Ct values detected with the more concentrated samples. Similar results were obtained using the E gene kit and reflected in the LamPORE and digital droplet experiments. We saw little amplification with LAMP testing, however as demonstrated via the RNA extraction results on a fragment analyser, RNA quality was poor and it is likely that passage through the extraction kit led to excessive template degradation. Commercial stool kits for LAMP testing in veterinary applications, suggesting further optimisation is needed for use on human stool samples.\n\nThe potential for faeco-oral asymptomatic transmission of SARS-CoV-2 has significant implications for FMT programmes globally. With FMT services on pause, treatment of patients with recurrent and refractory CDI remain sub-optimal with a likely consequential impact on morbidity, mortality and health care resource. Availability of a validated SARS-CoV-2 stool assay for donor screening would facilitate safe restart of FMT production and supply. Through our optimised methodology we have shown that using the VIASURE assay we can detect SARS-CoV-2 in stool samples containing more than 200 viral copies per gram. This is comparable to the widely used nasopharyngeal swab testing14. As faecal viral shedding persists long after clearance from the upper respiratory tract (for up to 33 days in stool), the direct testing of faecal samples arguably is necessary. It is notable that in the spiking experiments whereas RNA was detectable in lysate spiked samples in neither donor stool nor prepared FMT was directly spiked RNA detectable. This is probably due to RNA destruction during the extraction process whereas in lysate the genetic material is protected within virions. Furthermore, for the first time, in the current work we have been able to demonstrate the efficacy of a Nanopore technology platform in faecal samples. This exciting development opens the possibility of rapid near-subject testing of stool samples with potential for applications beyond the current COVID-19 pandemic.\n\nOur limited patient data demonstrates that about 40% of symptomatic patients admitted with acute COVID-19 test positive for SARS-CoV-2 in faecal samples and this is congruent with other published data6. Although the main impetus behind our work is to ensure that we have a suitable stool assay to resume our national FMT service, the finding of SARS-CoV-2 in stool highlights the intriguing potential role of the gastro-intestinal tract in the pathogenesis of the disease. We also have shown that SARS-CoV-2 cannot be detected in the stool of asymptomatic patients presenting with acute surgical emergencies in an area with high incidence of SARS-CoV-2. While true faecal-oral transmission and infectivity of stool samples from COVID-19 patients has yet to be confirmed, there may be sufficient circumstantial evidence to suggest that this is likely to be the case in certain circumstances.\n\nIn previous work examining antibody development against SARS-CoV2 about a quarter of hospital staff had diarrhoea and there was an association between this and the likelihood of antibody detection in serum18. There is accumulating evidence that the gut mounts an active immunoglobulin response to the virus19 and it is suggested that the gut IgA response may impact on the efficacy of the body’s response to vaccination. In vitro work from several laboratories have shown that intestinal epithelium is readily infected suggesting that the intestine is a potential site of SARS-CoV-2 replication20 with upregulation of viral response genes. Moreover, there is increasing evidence that SARS-CoV2 infection is associated with intestinal inflammation based on measurement of faecal calprotectin21. In contrast, a large retrospective cohort reported an intriguing suggestion that gastro-intestinal involvement may be associated with a more benign outcome of infection22. Although these data are subject to question in light of the retrospective study design there was a signal of a comparatively ‘anti-inflammatory’ peripheral cytokine response in patients with gastrointestinal symptoms.\n\nInterestingly, in our study of asymptomatic patients presenting to an acute surgical unit during the peak of the 2nd wave of SARS-CoV-2 infection in the UK, we found no positivity either via NP swab or in stool, despite a proportion of them having diarrhoea on admission. A single patient was identified who was previously positive for SARS-CoV-2 who had detectable virus in their stool 25 days after initial positivity. Together these findings suggest that faecal transmission in the absence of a positive nasopharyngeal swab is unlikely to be an issue, and that faecal transmission is not an important route in SARS-CoV-2 in asymptomatic patients.\n\nThe practice of FMT, while revolutionary for the treatment of CDI9 and showing promise in IBD23, has recently been stalled as a result of transmission of infective pathogens to patients who came to harm as a result10. Recent cases include drug resistant Escherichia coli (Enteropathogenic Escherichia coli and Shigatoxin-producing Escherichia coli) and related to inadequate screening of donated stool samples.\n\nIt is therefore imperative that donated FMT stool samples are carefully screened for SARS-CoV-2 and only used once tested negative12. The Chinese University of Hong Kong has an active FMT program and, similar to our approach, they have adopted a similar approach of careful enhanced donor screening and PCR testing of stool samples in order to perform FMT safely24.\n\n\nData availability\n\nHarvard Dataverse: RT-QPCR and LamPORE sequencing of spiked human faecal transplantation samples, https://doi.org/10.5061/dryad.70rxwdbx925.\n\nThis project contains the following underlying data:\n\n- SARS-CoV-2_Faecal_spiking_validation.xlsx (Excel spreadsheet giving overall results of project)\n\n- FTM-E_Gene_Check.eds (Ct values for E genes for project)\n\n- Ct_Values_FTM_-_Viasure_qPCR.csv\n\n- Ct_Values_FTM_-_Viasure_qPCR.csv (Ct values for ORF1ab and N for project)\n\n- CF06BB31-FMT-RNA-Viasure.eds (Thermofisher raw data for Lyaste & RNA spiked into FMT)\n\n- FMT-SManzoor_Lampore_data.zip (LamPORE sequencing data files)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nA previous version of this article is available on medRxiv: https://doi.org/10.1101/2021.02.02.21250934\n\n\nReferences\n\nZhang JJ, Dong X, Cao YY, et al.: Clinical characteristics of 140 patients infected with SARS-CoV-2 in Wuhan, China. 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PubMed Abstract \| Publisher Full Text\n\nGreen CA, Quraishi MN, Shabir S, et al.: Screening faecal microbiota transplant donors for SARS-CoV-2 by molecular testing of stool is the safest way forward. Lancet Gastroenterol Hepatol. 2020; 5(6): 531. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIaniro G, Mullish BH, Kelly CR, et al.: Reorganisation of faecal microbiota transplant services during the COVID-19 pandemic. Gut. 2020; 69(9): 1555–63. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCerTest: VIASURE SARS-CoV-2 Real Time PCR Detection Kit. 2020. Reference Source\n\nCorman VM, Landt O, Kaiser M, et al.: Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill. 2020; 25(3): 2000045. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBosworth A, Whalley C, Poxon C, et al.: Rapid implementation and validation of a cold-chain free SARS-CoV-2 diagnostic testing workflow to support surge capacity. 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[dataset]. 2021. http://www.doi.org/10.5061/dryad.70rxwdbx9" }	[ { "id": "89087", "date": "20 Jul 2021", "name": "Jonathan Landy", "expertise": [ "Reviewer Expertise FMT", "microbiota", "gut immunology", "IBD" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nFMT services are essential for patients with refractory or recurrent Clostridium difficile. The availability of FMT services during the COVID-19 pandemic has been problematic due to concerns regarding safety and screening for SARS-COV-2 in donor stool. Although no confirmed evidence of faecal-oral transmission is available, viral shedding in the GI tract has been established up to 5 weeks after symptom onset and the potential for transmission from donor stool during the pandemic theoretically exists. There is a need to ensure safety of FMT samples during the COVID-19 pandemic.\n\nThis report examines preparation methods and assays for detection of SAR-CoV-2 RNA in stool that might be employed in screening of donor stool to enable safe resumption of FMT services. Due to RNA degradation lysate spiked samples were used to assess detection at varying dilutions demonstrating the validity of the assays. Furthermore, assays were confirmed in symptomatic COVID-19 patients and in a swab positive asymptomatic patient.\n\nAlthough there may be concern regarding RNA degradation in preparation and homogenation of samples, virus can be detected in stool. However, in lower titres the assays did not detect viral RNA. This may be of concern in asymptomatic individuals with negative nasopharyngeal swabs where titres may be present, but below the limit of detection.\n\nThis report demonstrates the feasibility of detection of SARS-CoV-2 in screening of donor stool. However, uncertainty remains regarding the utility of this over repeated nasopharyngeal testing and exclusion of positive cases for a prolonged period and further data on current donor samples and recipients' will be important as FMT services resume.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "89234", "date": "21 Jul 2021", "name": "James Kinross", "expertise": [ "Reviewer Expertise Gut microbiome", "colorectal surgery and COVID19", "cancer." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nFMT is a highly effective intervention in the treatment of CDI, and it is of significant importance that this can continue to be administered to patients during all phases of the pandemic. Moreover, the gut represents and important mediator of the inflammatory response to COVID-19 and it is a potential source of transmission.\n\nThis analysis has attempted to create a novel assay for the detection of SARS-Cov-2 in faecal samples based on RT-qPCR and it has validated LAMP, LamPORE and droplet digital PCR for SARS-CoV-2 RNA detection. The study has applied an in-vitro analysis and validated these data in a prospective cohort of patients attending a UK NHS hospital.\nThis is a well written and well performed analysis. There is a grammatical error in the abstract conclusion. This should read (I assume..) \"SARS-CoV-2 can be detected...\".\n\nThe methodology is appropriate and well performed. The performance of the respective assays is reported.\n\nThe essential conclusion of this method development paper is that FMT samples could be safely tested for SARS-CoV-2 using RT-PCR or LamPORE. This adds weight to the concept that FMT services could in theory be safely re-started if these screening approaches were adopted. However, it is not possible to draw significant clinical inferences from this data set or to make comment on transmissibility of SARS-CoV-2 from faecal samples.\n\nI note that some of the symptomatic patients in this cohort were treated with Remdesivir, and none of these patients had detectable virus in the samples. Those with detectable viral load had some missing treatment data, but largely had either oxygen, antibiotics and in one case steroids. Therefore it is possible that there is some selection bias.\n\nThe clinical validation component of this study was not powered to an endpoint, but rather served as a pilot data set to demonstrate the performance of the assays under real world conditions.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-373
https://f1000research.com/articles/9-537/v1	05 Jun 20	{ "type": "Research Article", "title": "Human umbilical cord blood-mesenchymal stem cell-derived secretome in combination with atorvastatin enhances endothelial progenitor cells proliferation and migration", "authors": [ "Yudi Her Oktaviono", "Suryo Ardi Hutomo", "Makhyan Jibril Al-Farabi", "Angliana Chouw", "Ferry Sandra", "Suryo Ardi Hutomo", "Makhyan Jibril Al-Farabi", "Angliana Chouw", "Ferry Sandra" ], "abstract": "Background: Human umbilical cord blood-mesenchymal stem cell (hUCB-MSC)-derived secretome is known to be able to promote neovascularization and angiogenesis, so it is also thought to have a capability to modulate endothelial progenitor cell (EPC) functions. Atorvastatin is the cornerstone of coronary artery disease (CAD) treatment which can enhance EPCs proliferation and migration. This study aims to analyze the effect of the hUCB-MSC-derived secretome and its combination with atorvastatin toward EPCs proliferation and migration. Methods: EPCs were isolated from a CAD patient’s peripheral blood. Cultured EPCs were divided into a control group and treatment group of 2.5 µM atorvastatin, hUCB-MSC-derived secretome (2%, 10%, and 20% concentration) and its combination. EPCs proliferation was evaluated using an MTT cell proliferation assay, and EPC migration was evaluated using a Transwell migration assay kit. Results: This research showed that hUCB-MSC-derived secretomes significantly increase EPC proliferation and migration in a dose-dependent manner. The high concentration of hUCB-MSC-derived secretome were shown to be superior to atorvastatin in inducing EPC proliferation and migration (p<0.001). A combination of the hUCB-MSC-derived secretome and atorvastatin shown to improve EPCs proliferation and migration compared to hUCB-MSC-derived secretome treatment or atorvastatin alone (p<0.001). Conclusions: This study concluded that the hUCB-MSC-derived secretome work synergistically with atorvastatin treatment in improving EPCs proliferation and migration.", "keywords": [ "coronary artery disease", "endothelial progenitor cells", "mesenchymal stem cells", "secretome", "statins" ], "content": "Introduction\n\nCoronary artery disease (CAD) is the leading cause of mortality and morbidity worldwide1. It is responsible for the deaths of 7.2 million people or 12.2% of total deaths per year worldwide. Despite advancement in CAD management (e.g. novel antiplatelet therapy, coronary stents, percutaneous coronary intervention techniques and devices, and coronary artery bypass surgery), there are some clinical subsets of CAD which remain untreatable such as ischemic cardiomyopathy, refractory angina, and patient which cannot undergo revascularization due to clinical and anatomical complexity2,3.\n\nIt is already known that CAD is caused by atherosclerosis, which is followed by reduced levels of circulating endothelial progenitor cells (EPCs)4. EPCs can differentiate into mature endothelial cells and also promote endothelial repair. Hence, increasing circulating EPC levels is proven to improve endothelial function5. EPCs also had a critical role in the stimulation of angiogenesis and vasculogenesis. Hence, increasing EPC proliferation and migration may reduce ischemia and improve myocardial performance4,6.\n\nRegenerative treatment for CAD using stem cells has been extensively studied in the last decade7. However, these treatments faced challenges of low engraftment, poor survival, and low differentiation of the transplanted cells. Despite regenerative treatment shown to be promising in vitro, clinical studies showed unsatisfying results8. Hence, the researcher started to shift regenerative treatment from cell based-treatment into cell-free treatment using paracrine stimulation9. Nowadays, the usage of cell-free therapeutics as a regenerative therapy in cardiovascular diseases also started to be emerged9.\n\nThe secretome is the wide array variety of paracrine factors produced by mesenchymal stem cells (MSCs). Human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) derived secretome was proven could promote neovascularization, angiogenesis10–13 and improved cardiac systolic function by protecting myocardial cells from apoptosis14. However, using this approach to improve neovascularization is yet to be investigated. Hence, it is hypothesized that increasing EPCs proliferation and migration by the hUCB-MSC-derived secretome may be responsible for this effect. Statins, through their pleiotropic effect, are the cornerstone of CAD treatment. Atorvastatin is one of the most prescribed statins, whose ability to modulate EPCs proliferation and migration has already been well studied in both laboratory and clinical settings15–17. Furthermore, this study aims to compare the effect of the hUCB-MSC-derived secretome, atorvastatin and the two in combination in modulating EPC proliferation and migration.\n\n\nMethods\n\nA 50–100 mL peripheral blood sample was obtained from a patient with CAD. The patient was recruited from the outpatient cardiovascular clinic at Pusat Pelayanan Jantung Terpadu, Dr. Soetomo General Hospital, Surabaya, in March 2020. The inclusion criteria were as follows: male, aged 40–59 years old, history of chronic ischemic heart disease as proven by coronary angiography results that showed >50% stenosis of left main coronary artery or >70% of other coronary arteries. The exclusion criteria were as follows: a history of percutaneous coronary intervention procedures or coronary artery bypass grafting surgery, acute coronary syndromes, and anemia.\n\nThis study protocol has an ethical clearance from the Health Research Ethics Committee of Dr. Soetomo General Hospital, Surabaya (No.1567/KEPK/X/2019, approved on 8 October 2019). The included subjects provided written informed consent before subject recruitment. All details which include personal information were omitted.\n\nThe HUCB-MSCs-derived secretome was prepared according to the previous study18. HUCB-MSCs (3H Biomedical AB, Uppsala, Sweden) was cultured in MesenCultTM MSC Basal medium, supplemented with MesenCultTM Stimulatory supplement (StemCell Technologies Inc., Vancouver, Canada), and also added with penicillin and streptomycin. Upon reaching 80% confluency, the media was replaced with MesenCultTM MSC Basal medium (supplement-free media) and incubated for 24 hours. The media was collected and centrifuged. The supernatant was used as a conditioned medium that contained hUCB-MSCs-derived secretome.\n\nPeripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation of CAD patient’s peripheral blood using Histopaque-1077 (Sigma-Aldrich, USA). After centrifugation of peripheral blood, PBMCs then cultured with STEMLINE-II hematopoietic stem cell expansion medium (Sigma-Aldrich, USA) supplemented with stem cell factor, thrombopoietin, Flt-3 ligand, vascular endothelial growth factor, and interleukin-6. A total of 5×106 mononuclear cells/ml were seeded into fibronectin-coated 6-well plate dish and cultured at 37°C and 5% CO2 levels for 5 days. Non-adherent cells were then transferred for the proliferation and migration assay. After five days of culture, EPCs were confirmed using FITC-labeled anti-human CD34 antibody (animal source was mouse, 5 μL antibody was diluted at 500 μL per 1 × 106 cells; catalog number 60013FI, Gene ID: 947, by StemCell Technologies Inc., Vancouver, Canada) staining and examined with immunofluorescence microscopy.\n\nCultured EPCs were divided into eight treatment groups for each proliferation and migration assays. Those treatment include control group, 2.5 µM atorvastatin, low (2%), medium (10%) and high (20%) doses of hUCB-MSC-derived secretome, and combination of 2.5 µM atorvastatin with each dose of the hUCB-MSC-derived secretome. There were n=5 replications made from each treatment.\n\nThe MTT cell proliferation assay kit (Sigma-Aldrich, St Louis, MO, USA) was used to measure EPCs proliferation as described previously19. Treated EPCs were added with MTT reagent and incubated in a 37°C incubator with 5% CO2 for 4 hours. Proliferation was determined from the reduction of tetrazolium (MTT) into insoluble formazan product by viable EPCs mitochondria. Absorbance was measured with a microplate reader at 595 nm wavelength. EPCs proliferation was measured as optical density (OD). MTT assay was measured at day 3 after reagent addition.\n\nEPCs migration was evaluated using the 24-mm diameter insert, 3-µm pore size, 6-well Transwell migration assay kit (Corning, USA). A total of 5×105 cultured EPCs were placed in the upper part of the Transwell migration assay kit. Next, 2 mL of EPC media and each treatment were added in the lower chamber compartment and then incubated for 24 hours at 37°C. Non-migratory cells were removed manually. On the receiver plate, the new basal medium was placed and added 500 μL of trypsin + EDTA solution 0.5%, followed by 10 minutes incubation. Then, cells on the bottom surface of the membrane were stained with Giemsa and cell images were obtained on a light microscope and counted manually in n=5 random fields/sample.\n\nStatistical analyses were conducted using SPSS Statistics 23.0 to detect significance level at p<0.05. One-way ANOVA was used to compare groups, with Fisher’s least significant difference (LSD) post hoc test. Kruskal-Wallis test was used if there are violations to the assumption of normality and the assumption of homogeneity of variance. Correlation between variables was obtained using Spearman’s correlation followed by a linear regression test.\n\n\nResults\n\nClinical examination, blood sampling, electrocardiography, echocardiography and coronary angiography was conducted and evaluated in order to examine the inclusion and exclusion criteria. Our sample had a 1-year history of coronary artery disease, he suffered from refractory chest pain despite the optimum medical therapy. The coronary angiography showed complex lesion (three-vessel disease with chronic total occlusion) which was not amenable to undergo revascularization. The baseline characteristics of the patient are presented in Table 1.\n\nEPCs were succesfully isolated and cultured from the CAD patient’s peripheral blood. It was confirmed through light microscopy and an immunofluorescence assay (Figure 1). Raw images are available as Underlying data20.\n\n(A) DAPI staining of cultured EPCs showed viable cells through blue fluorescent of cells nuclei. (B) EPCs were confirmed, using FITC-labeled anti-human CD34 expression on immunofluorescence microscope. (C) Merged view of DAPI and FITC stained cells. (D) The light microscope view showed the spindle shape of EPCs. The white bar represents 50 µm.\n\nEPCs were evaluated using the MTT proliferation assay. As shown in Figure 2, both atorvastatin and hUCB-MSCs-derived secretome treatment groups at all doses increase EPCs proliferation compared to the control (p<0.05, ANOVA). hUCB-MSC-derived secretome treatment showed a dose-dependent relationship with EPCs proliferation. At medium (10%) and high (20%) doses, hUCB-MSC-derived secretome was shown to elicit superior EPC proliferation than atorvastatin (OD 1.252±0.104 and 1.585±0.029, respectively, vs 0.738±0.025; p<0.01). Raw absorbance data for MTT assays are available as Underlying data20.\n\nPearson’s correlation showed a significant and strong correlation between hUCB-MSCs-derived secretome treatment with EPC proliferation (r=0.954; p<0.001). The linear regression test showed an R2 of 0.910.\n\nFigure 2 showed the combination of atorvastatin and hUCB-MSC-derived secretome at the dose of 2%, 10% and 20% concentration have significantly higher EPCs proliferation compared to atorvastatin alone (OD 0.803±0.046, 1.298±0.075 and 1.761±0.419 vs 0.738±0.025, p<0.05). In addition, combination of hUCB-MSC-derived secretome at dose of 2%, 10% and 20% with atorvastatin showed higher EPCs proliferation compared to hUCB-MSC-derived secretome alone (OD 0.803±0.046 vs 0.713±0.049, 1.298±0.075 vs 1.252±0.104 and 1.761±0.419 vs 1.585±0.029, p<0.05). The combination group showed a significant and very strong correlation with EPC proliferation (r=0.973; p<0.001), Linear regression test showed R2 of 0.947.\n\naSignificant difference compared to the control group (p < 0.001). bSignificant difference compared to the 2.5 µM atorvastatin group (p < 0.001). cSignificant difference compared to the 2% hUCB-MSC-derived secretome group, (p <0.001). dSignificant difference compared to the 10% hUCB-MSC-derived secretome group (p < 0.001). eSignificant difference compared to the 20% hUCB-MSC-derived secretome group (p < 0.001), fSignificant difference compared to the combination of 2% hUCB-MSC-derived secretome and atorvastatin group, (p < 0.001), gSignificant difference compared to the combination of 10% hUCB-MSC-derived secretome and atorvastatin group, (p < 0.001). hSignificant difference compared to the combination of 20% hUCB-MSC-derived secretome and atorvastatin group, (p < 0.001)\n\nEPCs migration from each treatment group was analyzed using the Transwell migration assay. As shown in Figure 3, EPC treatment with atorvastatin and all doses of hUCB-MSCs-derived secretome significantly increase EPC migration compared to the control group (p<0.05, ANOVA). Treatment with 2.5 µM atorvastatin has significantly higher EPCs migration than low (2%) and medium (10%) doses of hUCB-MSC-derived secretome (34.40±3.05 vs 17.20±1.92 and 27.00±4.00, p<0.05). However, high doses (20%) of hUCB-MSC-derived secretome showed significantly higher migrated EPCs than atorvastatin (51.00±5.15 vs 34.40±3.05, p<0.001). Raw cell counts used to assess migration are available as Underlying data20.\n\nPearson’s correlation showed a significant and very strong correlation between hUCB-MSCs-derived secretome treatment with EPC migration (r=0.968; p<0.001). The linear regression test showed an R2 of 0.937.\n\nIn Figure 3, EPCs migration was significantly higher in combination treatment groups (atorvastatin and hUCB-MSC-derived secretome) at 2%, 10%, and 20% doses compared to the atorvastatin alone (38.20±3.49, 50.20±5.31 and 76.40±7.50 vs 34.40±3.05, p<0.001).\n\naSignificant difference compared to the control group (p < 0.001). bSignificant difference compared to the 2.5 µM atorvastatin group (p < 0.001). cSignificant difference compared to the 2% hUCB-MSC-derived secretome group (p < 0.001). dSignificant difference compared to the 10% hUCB-MSC-derived secretome group (p < 0.001). eSignificant difference compared to the 20% hUCB-MSC-derived secretome group (p < 0.001). fSignificant difference compared to the combination of 2% hUCB-MSC-derived secretome and atorvastatin group, (p < 0.001). gSignificant difference compared to the combination of 10% hUCB-MSC-derived secretome and atorvastatin group, (p < 0.001). hSignificant difference compared to the combination of 20% hUCB-MSC-derived secretome and atorvastatin group, (p < 0.001)\n\nCombination of hUCB-MSC-derived secretome with atorvastatin also showed higher EPCs migration than the hUCB-MSC-derived secretome-only group at 2%, 10% and 20% concentrations (38.3±3.49 vs 17.20±1.92, 50.20±5.31 vs 27.00±4.00 and 76.40±7.50 vs 51.00±5.15, respectively; all p<0.001). The combination of high-dose (20%) hUCB-MSC-derived secretome and atorvastatin had the highest number of migrated EPC (76.4±7.50 × 103 cells). The combination group had a significant and very strong correlation with EPC migration (r=0.970; p<0.001). The linear regression test showed an R2 of 0.942.\n\n\nDiscussion\n\nThis research showed that treatment with hUCB-MSC-derived secretome, atorvastatin and a combination of the two increased the proliferation and migration of EPCs (isolated from CAD patient’s peripheral blood). HUCB-MSC-derived secretome enhances EPCs proliferation and migration in a dose-dependent manner. The combination of hUCB-MSC-derived secretome and atorvastatin was shown to be superior to atorvastatin or hUCB-MSC-derived secretome alone.\n\nIn this research, hUCB-MSC-derived secretome treatment increased EPC proliferation in a dose-dependent manner, with the concentrations of 10% and 20% shown to be superior to atorvastatin. Previous studies showed that atorvastatin treatment is superior to other statins at improving EPC proliferation21–23. The HUCB-MSC-derived secretome is also composed of cytokines, chemokines, growth factors, proteins, and extracellular vesicles which may be involved in EPCs proliferation and migration13,24. Vascular endothelial growth factor (VEGF), stromal-derived factor-1 (SDF-1), insulin-like growth factor (IGF-1) are contained in the hUCB-MSC-derived secretome which may be involved in increasing EPC proliferation25. VEGF has been shown to improve the proliferation and differentiation of EPCs through activation of Ras signaling, and the MAPK/ERK pathway26–28. SDF-1 and IGF 1 also been shown to increase the EPCs proliferation in response to the PI3K/protein kinase B signaling pathway and promote angiogenesis29–31. Hence, it is suggested that hUCB-MSC-derived secretome treatment is beneficial to improve EPC proliferation which may involve MAPK/ERK and PI3K/protein kinase B pathway.\n\nHUCB-MSC-derived secretome treatment shown to increase EPCs migration in a dose-dependent manner, with a concentration of 20% shown to be superior to atorvastatin. Similarly, the previous study showed secretome-derived from placental-MSCs is able to significantly increase EPCs migration32. The HUCB-MSC-derived secretome contains pro-angiogenic factors, such as human angiopoietin-1 (Ang-1), hepatocyte growth factor (HGF), insulin-like growth factor I (IGF-I), prostaglandin E2 (PGE2), transforming growth factor-beta 1 (TGF-ß1), vascular cell adhesion protein 1 (VCAM-1) and vascular endothelial growth factor (VEGF)33. MSCs also have immunomodulatory and anti-inflammatory properties, as it contributes to the maintenance of self-renewal capacity through E-Prostanoid 2 (EP2)34 and immune cell activation and maturation, including CD4+ helper T cells, B cells, dendritic cells, natural killer cells, monocytes and macrophages35. The HUCB-MSC-derived secretome also has a higher anti-inflammatory effect than other MSCs36 and antioxidant properties, as proven in previous studies conducted in renal injury37 and ischemic stroke38. Inflammatory stimuli and oxidative stress are also known to impair EPCs migration39. Chemoattractant gradient was an important driving factor to induce EPCs mobilization. Hence, a high concentration of growth factors in the HUCB-MSC-derived secretome increase the gradient between the top and lower parts of the Transwell migration assay may lead into an increase of EPCs migration. taken together, wide array molecules and multiple possible pathways involved in HUCB-MSCs secretome treatment seem to be responsible for its superiority against atorvastatin.\n\nThe synergistic effect of the HUCB-MSC-derived secretome with atorvastatin in enhancing EPCs proliferation and migration was demonstrated in this study. These combinations significantly increase EPCs proliferation and migratory activity by up to two-fold. Previously, The combination of MSCs with another compound, including statins, was shown to have beneficial effects in angiogenesis and neovascularization40–42. Co-culture of MSCs and EPCs have been shown to demonstrate improved EPC proliferation and migration, and enhance their angiogenic capacity43,44. However, the exact mechanism of these combinations to improve EPCs proliferation and migration is yet to be investigated. It is speculated that the involvement of multiple pathways may be responsible for its superiority against HUCB-MSCs-derived secretome or atorvastatin alone.\n\nA mitogen-activated protein kinase (MAPK) pathway has been known to play a role in increasing EPCs proliferation45. Cell cycle progression through increased Cyclin D1 expression mediated by PI3K/Akt and MAPK pathway also involved in EPCs proliferation46. While increasing microRNA 221/222 expression shown to reduce EPCs proliferation capabilities47. HUCB-MSC-derived secretome treatment was speculated to improve EPCs proliferation through MAPK/ERK and PI3K/protein kinase B pathway. While atorvastatin improves EPCs proliferation through downregulation of microRNA 221/222 expression47. The involvement of these multiple pathways may result in higher EPCs proliferation in the combination group.\n\nEnhanced growth factor levels through hUCB-MSC-derived secretome treatment will augment the chemoattractant gradient37, thus leading to enhanced migration of EPCs. The anti-inflammatory and antioxidant properties of hUCB-MSC-derived secretome also speculated to improve EPCs migration35,37. While atorvastatin can increase the production of endothelial nitric oxide synthase and nitric oxide, which reduces the oxidative stress that impairs EPCs migration42. Statin also could prevent EPCs senescence by upregulating TRF2 of EPCs, hence enhance migratory capacity48. Those facts suggested that the combination of hUCB-MSC-derived secretome and atorvastatin will have superior EPCs migration through the involvement of multiple pathways.\n\nIn summary, hUCB-MSC-derived secretome may be developed and combined with atorvastatin treatment in CAD patients to improve EPCs proliferation and migration. However, this research did not measure the exact composition of the hUCB-MSC-derived secretome. The previous study showed that the secretome from another type of MSC can increase EPCs migration but not EPCs proliferation49. Hence, further research should be directed to identify the substance within the hUCB-MSC-derived secretome which is responsible for increasing EPC proliferation and migration, and compare it with other MSC secretomes. Further research should also verify the multiple pathways which may be responsible for the improvement of EPCs proliferation and migration in the combination group.\n\n\nConclusions\n\nHigh dose hUCB-MSC-derived secretome outperforms atorvastatin to improve EPC proliferation and migration. A combination of hUCB-MSC-derived secretome with atorvastatin seems to be beneficial in promoting neovascularization through improved EPCs proliferation and migration effect compared to hUCB-MSC-derived secretome or atorvastatin alone.\n\n\nData availability\n\nFigshare: Human umbilical cord blood-mesenchymal stem cell-derived secretome in combination with atorvastatin enhances endothelial progenitor cells proliferation and migration. https://doi.org/10.6084/m9.figshare.12186507.v220.\n\nThis project contains the following underlying data:\n\nRAW Data - F1000 revisi2 by SAH (XLSX). (Raw absorbance data from MTT proliferation assay and cell counts from the Transwell migration assay.)\n\nRAW DATA f1000 (ZIP). (Raw images generated in this study, including images used to generate cell counts and raw immunofluorescence images.)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nThe authors are thankful to Christian Pramudita, MD., Ilma Alfia Isaridha, MD., Melly Susanti MD., and Dwi Fachrul MD., for invaluable support in this project.\n\n\nReferences\n\nBenjamin EJ, Muntner P, Alonso A, et al.: Heart Disease and Stroke Statistics-2019 Update: A Report From the American Heart Association. Circulation. 2019; 139(10): e56–e528. PubMed Abstract \| Publisher Full Text\n\nSainsbury PA, Fisher M, de Silva R: Alternative interventions for refractory angina. Heart. 2017; 103(23): 1911–1922. 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Publisher Full Text\n\nOktaviono YH, Al-Farabi MJ, Oliva L, et al.: Preliminary Study: Purple Sweet Potato Extract Seems to Be Superior to Increase the Migration of Impaired Endothelial Progenitor Cells Compared to l -Ascorbic Acid. Sci Pharm. 2017; 87(3): 16. Publisher Full Text\n\nAl Farabi, Makhyan JibrilHuman umbilical cord blood-mesenchymal stem cell-derived secretome in combination with atorvastatin enhances endothelial progenitor cells proliferation and migration. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12186507.v2\n\nMeuthia F, Oktaviono YH, Soemantri D: Effects of Statins on Endothelial Progenitor Cell Proliferation from Peripheral Blood of Stable Coronary Artery Disease Patient. J Kardiol Indons. 2017; 38(1): 6–12. Publisher Full Text\n\nOktaviono YH, Savitri TVR, Soemantri D: Rosuvastatin is Superior Compared to Simvastatin and Atorvastatin to Induce Endothelial Progenitor Cells Migration. J Clin Diagnostic Res. 2019; 1077: 5–8. Publisher Full Text\n\nRawlings R, Nohria A, Liu PY, et al.: Comparison of effects of rosuvastatin (10 mg) versus atorvastatin (40 mg) on rho kinase activity in caucasian men with a previous atherosclerotic event. Am J Cardiol. 2009; 103(4): 437–441. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEleuteri S, Fierabracci A: Insights Into the Secretome of Mesenchymal Stem Cells and Its Potential Applications. Int J Mol Sci. 2019; 20(18): 4597. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKwon HM, Hur SM, Park KY, et al.: Multiple paracrine factors secreted by mesenchymal stem cells contribute to angiogenesis. Vascul Pharmacol. 2014; 63(1): 19–28. PubMed Abstract \| Publisher Full Text\n\nXu J, Liu X, Jiang Y, et al.: MAPK/ERK signalling mediates VEGF-induced bone marrow stem cell differentiation into endothelial cell. J Cell Mol Med. 2008; 12(6A): 2395–2406. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGe Q, Zhang H, Hou J, et al.: VEGF secreted by mesenchymal stem cells mediates the differentiation of endothelial progenitor cells into endothelial cells via paracrine mechanisms. Mol Med Rep. 2018; 17(1): 1667–1675. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nArutyunyan I, Fatkhudinov T, Kananykhina E, et al.: Role of VEGF-A in angiogenesis promoted by umbilical cord-derived mesenchymal stromal/stem cells: in vitro study. Stem Cell Res Ther. 2016; 7: 46. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen SL, Sheiban I, Xu BO, et al.: Impact of the complexity of bifurcation lesions treated with drug-eluting stents: the DEFINITION study (Definitions and impact of complEx biFurcation lesIons on clinical outcomes after percutaNeous coronary IntervenTIOn using drug-eluting steNts). JACC Cardiovasc Interv. 2014; 7(11): 1266–1276. PubMed Abstract \| Publisher Full Text\n\nYu H, Feng Y: The potential of statin and stromal cell-derived factor-1 to promote angiogenesis. Cell Adh Migr. 2008; 2(4): 254–257. Publisher Full Text\n\nHou J, Peng X, Wang J: Mesenchymal stem cells promote endothelial progenitor cell proliferation by secreting insulin-like growth factor-1. Mol Med Rep. 2017; 16(2): 1502–1508. PubMed Abstract \| Publisher Full Text\n\nKamprom W, Kheolamai P, U-Pratya Y: Endothelial Progenitor Cell Migration-Enhancing Factors in the Secretome of Placental-Derived Mesenchymal Stem Cells. Stem Cells Int. 2016; 2016: 2514326. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWu M, Zhang R, Zou Q, et al.: Comparison of the Biological Characteristics of Mesenchymal Stem Cells Derived from the Human Placenta and Umbilical Cord. Sci Rep. 2018; 8(1): 5014. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee BC, Kim HS, Shin TH, et al.: PGE 2 maintains self-renewal of human adult stem cells via EP2-mediated autocrine signaling and its production is regulated by cell-to-cell contact. Sci Rep. 2016; 6: 26298. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMurphy MB, Moncivais K, Caplan AI: Mesenchymal stem cells: Environmentally responsive therapeutics for regenerative medicine. Exp Mol Med. 2013; 45(11): e54. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJin HJ, Bae YK, Kim M, et al.: Comparative analysis of human mesenchymal stem cells from bone marrow, adipose tissue, and umbilical cord blood as sources of cell therapy. Int J Mol Sci. 2013; 14(9): 17986–18001. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee KH, Tseng WC, Yang CY, et al.: The Anti-Inflammatory, Anti-Oxidative, and Anti-Apoptotic Benefits of Stem Cells in Acute Ischemic Kidney Injury. Int J Mol Sci. 2019; 20(14): pii: E3529. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCunningham CJ, Redondo-castro E, Allan SM: The therapeutic potential of the mesenchymal stem cell secretome in ischaemic stroke. J Cereb Blood Flow Metab. 2018; 38(8): 1276–1292. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTousoulis D, Andreou I, Antoniades C, et al.: Role of inflammation and oxidative stress in endothelial progenitor cell function and mobilization: Therapeutic implications for cardiovascular diseases. Atherosclerosis. 2008; 201(2): 236–247. PubMed Abstract \| Publisher Full Text\n\nLuo R, Lu Y, Liu J, et al.: Enhancement of the efficacy of mesenchymal stem cells in the treatment of ischemic diseases. Biomed Pharmacother. 2019; 109(1): 2022–2034. PubMed Abstract \| Publisher Full Text\n\nKwon YW, Heo SC, Jeong GO, et al.: Tumor necrosis factor-α-activated mesenchymal stem cells promote endothelial progenitor cell homing and angiogenesis. Biochim Biophys Acta. 2013; 1832(12): 2136–2144. PubMed Abstract \| Publisher Full Text\n\nSong L, Yang YJ, Dong QT, et al.: Atorvastatin enhance efficacy of mesenchymal stem cells treatment for swine myocardial infarction via activation of nitric oxide synthase. PLoS One. 2013; 8(5): 1–12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiang T, Zhu L, Gao W, et al.: Coculture of endothelial progenitor cells and mesenchymal stem cells enhanced their proliferation and angiogenesis through PDGF and Notch signaling. FEBS Open Bio. 2017; 7: 1722–1736. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi Z, Yang A, Yin X, et al.: Mesenchymal stem cells promote endothelial progenitor cell migration, vascularization, and bone repair in tissue-engineered constructs via activating CXCR2-Src-PKL/Vav2-Rac1. FASEB J. 2014; 400(1–2): 201–206. PubMed Abstract \| Publisher Full Text\n\nSandra F, Oktaviono YH, Widodo MA: Endothelial progenitor cells proliferated via MEK-dependent p42 MAPK signaling pathway. Mol Cell Biochem. 2015; 400(1–2): 201–6. PubMed Abstract \| Publisher Full Text\n\nQiu C, Xie Q, Zhang D, et al.: GM-CSF induces cyclin D1 expression and proliferation of endothelial progenitor cells via PI3K and MAPK signaling. Cell Physiol Biochem. 2014; 63(1): 19–28. PubMed Abstract \| Publisher Full Text\n\nMinami Y, Satoh M, Maesawa C, et al.: Effect of atorvastatin on microRNA 221 / 222 expression in endothelial progenitor cells obtained from patients with coronary artery disease. Eur J Clin Invest. 2009; 39(5): 359–67. PubMed Abstract \| Publisher Full Text\n\nAssmus B, Urbich C, Aicher A, et al.: HMG-CoA reductase inhibitors reduce senescence and increase proliferation of endothelial progenitor cells via regulation of cell cycle regulatory genes. Circ Res. 2003; 92(9): 1049–55. PubMed Abstract \| Publisher Full Text\n\nKamprom W, Kheolamai P, U-Pratya Y, et al.: Effects of mesenchymal stem cell-derived cytokines on the functional properties of endothelial progenitor cells. Eur J Cell Biol. 2016; 95(3–5): 153–63. PubMed Abstract \| Publisher Full Text" }	[ { "id": "64524", "date": "08 Jul 2020", "name": "Anwar Santoso", "expertise": [ "Reviewer Expertise 1. Cardiovascular disease (particularly ischemic heart disease) 2. Lipidology and diabetes mellitus 3. Hypertension 4. Stem cell and regenerative medicine" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study aims to compare the effect of hUCB-MSC-derived secretome, atorvastatin, and the two combinations in modulating EPC proliferation and migration. The study addresses the novel issues in refractory angina, whether the atorvastatin and secretome derived mesenchymal stem cells improve EPC expression. There are similar studies available addressing these issues. Zhang X et al. demonstrated that intravenous transplantation of huC-MSCs at an early stage could improve hypoxic-ischemic rats' behavior and decreased gliosis, this study was measured in other target disorders1.\n\nThe rationale and scientific background of this manuscript were justified, to disclose the role of secretomes and paracrine stimulation on EPC expression.\n\nIn the #Method section, the authors mentioned the inclusion criteria: male, aged 40-59 years old, history of chronic ischemic heart disease as proven by CAG (coronary angiography). The authors should quote the diagnostic criterion. Additionally, the authors should clearly state this is an experimental study of atorvastatin and hUCB-MSC-derived secretome and their combinations on EPC proliferation and migration.\n\nUmbilical cord blood-derived EPC established in a previous study displayed cobblestone-like morphology; this is a typical feature of the EPC. The authors did not state this in the manuscript, except they confirmed using FITC-labelled anti-human CD 34+ expression. The authors should clearly explain it[ref-2].\n\nThere was another immunophenotype of EPC as determined by flow cytometry, VEGFR2-PE, vWF-FITC, and CD31-PE2. Is there any reason why the authors only demonstrated with CD34+ expression. The authors should provide their ideas on it.\n\nIn the #EPCs proliferation assay, the authors explain EPCs proliferation measured using OD; there was no explanation of how OD transferred in a measurement scale in Figure 2 (Y-ordinate)?\n\nHow did the authors determine the percentage of hUCB-MSC-derived secretome? Is there any control over the measurement?\n\nIn the #Table 1 – Characteristics of the patient, the authors should explain \"Left ventricle internal diameter\" = 5.8 cm; I wonder whether that is either \"end-systolic dimension or end-diastolic dimension\"?\n\nIn #summary, the authors should open the opportunity on the horizon, whether the cell-based therapy or cell-free measures that win the future game?3.\nAgain, I would express my appreciation to all authors to address these evolving issues in regenerative medicine.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5820", "date": "10 May 2021", "name": "Yudi Her Oktaviono", "role": "Author Response", "response": "1. Q The study aims to compare the effect of hUCB-MSC-derived secretome, atorvastatin, and the two combinations in modulating EPC proliferation and migration. The study addresses the novel issues in refractory angina, whether the atorvastatin and secretome derived mesenchymal stem cells improve EPC expression. There are similar studies available addressing these issues. Zhang X et al. demonstrated that intravenous transplantation of huC-MSCs at an early stage could improve hypoxic-ischemic rats' behavior and decreased gliosis, this study was measured in other target disorders1. A. Thank you for the references and comparison, we will note this feedback and put it on the paper as other research which did similar intervention 2. Q. The rationale and scientific background of this manuscript were justified, to disclose the role of secretomes and paracrine stimulation on EPC expression.I A. ndeed, that was our research aim 3. Q. In the #Method section, the authors mentioned the inclusion criteria: male, aged 40-59 years old, history of chronic ischemic heart disease as proven by CAG (coronary angiography). The authors should quote the diagnostic criterion. Additionally, the authors should clearly state this is an experimental study of atorvastatin and hUCB-MSC-derived secretome and their combinations on EPC proliferation and migration. A. We have added the diagnostic criteria of chronic ischemic heart disease as proven by coronary angiography results that showed >50% stenosis of left main coronary artery or >70% of other coronary arteries. We also explain that this is an experimental study of atorvastatin and hUCB-MSC-derived secretome and their combinations on EPC proliferation and migration. 4. Q. Umbilical cord blood-derived EPC established in a previous study displayed cobblestone-like morphology; this is a typical feature of the EPC. The authors did not state this in the manuscript, except they confirmed using FITC-labelled anti-human CD 34+ expression. The authors should clearly explain it[ref-2]. A. Thank you very much for reminding us, We did evaluate the EPCs cobblestone-like morphology before staining the cells with CD34 antibody. We already put these information on the method section. 5. Q. There was another immunophenotype of EPC as determined by flow cytometry, VEGFR2-PE, vWF-FITC, and CD31-PE2. Is there any reason why the authors only demonstrated with CD34+ expression. The authors should provide their ideas on it. A. In this study, the use of FITC CD34+ only to confirmed the EPCs are sufficient, as the same EPCs culture method was used in authors previous research ( which mentioned on references number 19, 21-22,45). There was also uncertainty about the use of another immunophenotype of EPC as determined by flow cytometry (VEGFR2-PE, vWF-FITC, and CD31-PE), caused by heterogenous types of EPCs (mentioned on the references of 51,52) 6. Q. In the #EPCs proliferation assay, the authors explain EPCs proliferation measured using OD; there was no explanation of how OD transferred in a measurement scale in Figure 2 (Y-ordinate)? A. EPCs proliferation can be determined through various method, In the previous study, proliferation can be determined relatively through OD (references no 21-22,45). Thus, we use OD as proliferation measurement scale in Figure 2 (Y-ordinate). 7. Q.How did the authors determine the percentage of hUCB-MSC-derived secretome? Is there any control over the measurement? A. The percentage of hUCB-MSC-derived secretome was determined from the dilution level of hUCB-MSC-derived secretome. For example, 1 mL of hUCB-MSC-derived secretome with 49 mL of the phosphate buffer saline (PBS) plus 2% fetal bovine serum (FBS). However, no control over measurement. 8. Q. In the #Table 1 – Characteristics of the patient, the authors should explain \"Left ventricle internal diameter\" = 5.8 cm; I wonder whether that is either \"end-systolic dimension or end-diastolic dimension\"? A. It should be Left ventricle end-diastolic dimension, thanks for correcting this phrase 9. Q. In #summary, the authors should open the opportunity on the horizon, whether the cell-based therapy or cell-free measures that win the future game? A. Cardiac cell-based therapy has emerged as a novel therapeutic option for patients dealing with untreatable refractory angina (RA). However, after more than a decade of controlled studies, no definitive consensus has been reached regarding clinical efficacy. While in this research hUCB-MSC-derived secretome may be developed and combined with atorvastatin treatment in CAD patients to improve EPCs proliferation and migration. In this early study, we can conclude that secretome-based treatment may be a game changer in refractory angina therapeutic options and outperforms the previous cell-based therapy. Again, I would express my appreciation to all authors to address these evolving issues in regenerative medicine." }, { "c_id": "6601", "date": "10 May 2021", "name": "Yudi Her Oktaviono", "role": "Author Response", "response": "1. Q The study aims to compare the effect of hUCB-MSC-derived secretome, atorvastatin, and the two combinations in modulating EPC proliferation and migration. The study addresses the novel issues in refractory angina, whether the atorvastatin and secretome derived mesenchymal stem cells improve EPC expression. There are similar studies available addressing these issues. Zhang X et al. demonstrated that intravenous transplantation of huC-MSCs at an early stage could improve hypoxic-ischemic rats' behaviour and decreased gliosis, this study was measured in other target disorders1. A. Thank you for the references and comparison, we will note this feedback and put it on the paper as for other research which did a similar intervention 2. Q. The rationale and scientific background of this manuscript were justified, to disclose the role of secretomes and paracrine stimulation on EPC expression A. Indeed, that was our research aim 3. Q. In the #Method section, the authors mentioned the inclusion criteria: male, aged 40-59 years old, history of chronic ischemic heart disease as proven by CAG (coronary angiography). The authors should quote the diagnostic criterion. Additionally, the authors should clearly state this is an experimental study of atorvastatin and hUCB-MSC-derived secretome and their combinations on EPC proliferation and migration. A. We have added the diagnostic criteria of chronic ischemic heart disease as proven by coronary angiography results that showed >50% stenosis of left main coronary artery or >70% of other coronary arteries. We also explain that this is an experimental study of atorvastatin and hUCB-MSC-derived secretome and their combinations on EPC proliferation and migration. 4. Q. Umbilical cord blood-derived EPC established in a previous study displayed cobblestone-like morphology; this is a typical feature of the EPC. The authors did not state this in the manuscript, except they confirmed using FITC-labelled anti-human CD 34+ expression. The authors should clearly explain it[ref-2]. A. Thank you very much for reminding us, We did evaluate the EPCs cobblestone-like morphology before staining the cells with CD34 antibody. We already put these information on the method section. 5. Q. There was another immunophenotype of EPC as determined by flow cytometry, VEGFR2-PE, vWF-FITC, and CD31-PE2. Is there any reason why the authors only demonstrated with CD34+ expression. The authors should provide their ideas on it. A. In this study, the use of FITC CD34+ only to confirmed the EPCs are sufficient, as the same EPCs culture method was used in authors previous research ( which mentioned on references number 19, 21-22,45). There was also uncertainty about the use of another immunophenotype of EPC as determined by flow cytometry (VEGFR2-PE, vWF-FITC, and CD31-PE), caused by heterogenous types of EPCs (mentioned on the references of 51,52) 6. Q. In the #EPCs proliferation assay, the authors explain EPCs proliferation measured using OD; there was no explanation of how OD transferred in a measurement scale in Figure 2 (Y-ordinate)? A. EPCs proliferation can be determined through various method, In the previous study, proliferation can be determined relatively through OD (references no 21-22,45). Thus, we use OD as proliferation measurement scale in Figure 2 (Y-ordinate). 7. Q.How did the authors determine the percentage of hUCB-MSC-derived secretome? Is there any control over the measurement? A. The percentage of hUCB-MSC-derived secretome was determined from the dilution level of hUCB-MSC-derived secretome. For example, 1 mL of hUCB-MSC-derived secretome with 49 mL of the phosphate buffer saline (PBS) plus 2% fetal bovine serum (FBS). However, no control over measurement. 8. Q. In the #Table 1 – Characteristics of the patient, the authors should explain \"Left ventricle internal diameter\" = 5.8 cm; I wonder whether that is either \"end-systolic dimension or end-diastolic dimension\"? A. It should be Left ventricle end-diastolic dimension, thanks for correcting this phrase 9. Q. In #summary, the authors should open the opportunity on the horizon, whether the cell-based therapy or cell-free measures that win the future game? A. Cardiac cell-based therapy has emerged as a novel therapeutic option for patients dealing with untreatable refractory angina (RA). However, after more than a decade of controlled studies, no definitive consensus has been reached regarding clinical efficacy. While in this research hUCB-MSC-derived secretome may be developed and combined with atorvastatin treatment in CAD patients to improve EPCs proliferation and migration. In this early study, we can conclude that secretome-based treatment may be a game changer in refractory angina therapeutic options and outperforms the previous cell-based therapy. Again, I would express my appreciation to all authors to address these evolving issues in regenerative medicine." } ] }, { "id": "64523", "date": "03 Sep 2020", "name": "John David Symmons", "expertise": [ "Reviewer Expertise Anwar Tandar", "MD: Clinician", "Interventional Cardiologist. John D. Symmons", "PhD: Basic Scientist" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting research involving the evaluation of EPCs in CAD and association with statin.\nSeveral concerns should be addressed:\nIt is derived from one patient. The conclusion and validity need to be addressed seriously when there is sample from one patient. In addition, since there is only one patient, why is there even a need for inclusion and exclusion criteria\n\nTypgraphical error in the Introduction... refractory angina, and patients\n\nReference needed for the statement: HUCB-MSCs-derived secretome preparation. The media was collected......(reference 3).\n\nTreatment of EPCs...Please describe the rationale for 5 replications. Please describe the rationale behind the selected number 5.4.\n\nAll abbreviations need to be spelled out in the beginning or first time used\n\nSimilar to the rational for EPCs treatment, in EPC migrations assay section: Please describe the rationale for 5 fields and describe the sizes of the field.\n\nThis interesting research should investigate more patients to allow a stronger interpretation and to allow more sound conclusion\n\nOther statins should also be investigated as they are being used widely in real world.\n\nThe exact composition of the hUCB-MSc derived secretome should be described.\nIn summary, this research needs more diligent work especially with the number of samples from more robust patients population.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "6158", "date": "10 May 2021", "name": "Yudi Her Oktaviono", "role": "Author Response", "response": "Dear Dr Anwar Tandar Thank you very much for your valuable feedback I am truly honoured with your feedback In this opportunity, please let me answer the question Q: It is derived from one patient. The conclusion and validity need to be addressed seriously when there is sample from one patient. In addition, since there is only one patient, why is there even a need for inclusion and exclusion criteria. A: In this experimental laboratory research, the sample used was EPCs derived from single patient with coronary artery disease. We have ensured the diagnosis of coronary artery disease from the ECG, echocardiography and coronary angiography. The purpose of the single sample being used in this research is to ensure the homogeneity of the sample while inclusion and exclusion criteria was used to identify the best EPCs quality that represent patient with CAD. Mixing EPCs from the various patient may cause EPCs failed to grow efficiently due to incompatibility with other source of EPCs. Inclusion and exclusion criteria is still relevant because we want to ensure that the patient was really diagnosed with coronary artery disease with specific criteria without any other significant disease. Without clear inclusion and exclusion criteria, we may select patient with multiple disease which EPCs function may differ from patient with coronary artery disease only. Q: Typographical error in the Introduction... refractory angina, and patients A: Thank you for the feedback, we will revise accordingly Q: Reference needed for the statement: HUCB-MSCs-derived secretome preparation. The media was collected......(reference 3). A: We have added the reference, thank you Q: Treatment of EPCs...Please describe the rationale for 5 replications. Please describe the rationale behind the selected number 5 A: The 5 replication was based on Federer's formula: (t-1)(n-1) ≥ 15, where t is the number of treatments and n is the number of replication. Q: All abbreviations need to be spelt out in the beginning or first time used A: Thank you for the feedback, we will revise accordingly Q: Similar to the rationale for EPCs treatment, in EPC migrations essay section: Please describe the rationale for 5 fields and describe the sizes of the field. A: The usage of the 5 fields was the standard protocol of cell migration calculation using transwell migration assay based on the Transwell protocol. In this research, we used 0.04mm in diameter as the sizes of the field. Q: This interesting research should investigate more patients to allow a stronger interpretation and to allow more sound conclusion A: Thank you very much for your genuine feedback. This research was part of our preliminary research which explore the possibilities of secretome as an alternative regenerative treatment other than cell-based regeneration treatment for the patient with coronary artery disease. In this early research we used limited patient but we will expand further the research with involving more samples and aim to have clinical trials should we achieve satisfying results. We also consider to combine the secretome treatment with existing coronary artery disease treatment, to ensure the usage of secretome combined with current medical treatment will benefit synergistically. Q: Other statins should also be investigated as they are being used widely in real world. A: Thank you for the feedback, we will consider that as our future research suggestion. However, we first prefer atorvastatin since it is readily available in our country, Indonesia. Previously, we have compared the effect of atorvastatin, rosuvastatin and simvastatin effect on the EPCs (without secretome) and conclude that atorvastatin was the most superior in inducing EPCs migration. Thus, this research prefers to use atorvastatin and combine it with secretome. Q: The exact composition of the hUCB-MSc derived secretome should be described. A: Thanks, we would like to admit that the limitation of this research was the inability to exactly describe the molecule in the hUCB-MSc derived secretome. Further research is required through proteomic analysis to determine the molecule in the hUCB-MSc derived secretome. This will also help to standardize the composition of hUCB-MSc derived secretome. Again, many thanks for the review,, Best Regards Yudi Her" }, { "c_id": "6604", "date": "10 May 2021", "name": "Yudi Her Oktaviono", "role": "Author Response", "response": "Dear Dr Anwar Tandar Thank you very much for your valuable feedback I am truly honoured with your feedback In this opportunity, please let me answer the question Q: It is derived from one patient. The conclusion and validity need to be addressed seriously when there is sample from one patient. In addition, since there is only one patient, why is there even a need for inclusion and exclusion criteria. A: In this experimental laboratory research, the sample used was EPCs derived from single patient with coronary artery disease. We have ensured the diagnosis of coronary artery disease from the ECG, echocardiography and coronary angiography. The purpose of the single sample being used in this research is to ensure the homogeneity of the sample while inclusion and exclusion criteria was used to identify the best EPCs quality that represent patient with CAD. Mixing EPCs from the various patient may cause EPCs failed to grow efficiently due to incompatibility with other source of EPCs. Inclusion and exclusion criteria is still relevant because we want to ensure that the patient was really diagnosed with coronary artery disease with specific criteria without any other significant disease. Without clear inclusion and exclusion criteria, we may select patient with multiple disease which EPCs function may differ from patient with coronary artery disease only. Q: Typographical error in the Introduction... refractory angina, and patients A: Thank you for the feedback, we will revise accordingly Q: Reference needed for the statement: HUCB-MSCs-derived secretome preparation. The media was collected......(reference 3). A: We have added the reference, thank you Q: Treatment of EPCs...Please describe the rationale for 5 replications. Please describe the rationale behind the selected number 5 A: The 5 replication was based on Federer's formula: (t-1)(n-1) ≥ 15, where t is the number of treatments and n is the number of replication. Q: All abbreviations need to be spelt out in the beginning or first time used A: Thank you for the feedback, we will revise accordingly Q: Similar to the rationale for EPCs treatment, in EPC migrations essay section: Please describe the rationale for 5 fields and describe the sizes of the field. A: The usage of the 5 fields was the standard protocol of cell migration calculation using transwell migration assay based on the Transwell protocol. In this research, we used 0.04mm in diameter as the sizes of the field. Q: This interesting research should investigate more patients to allow a stronger interpretation and to allow more sound conclusion A: Thank you very much for your genuine feedback. This research was part of our preliminary research which explore the possibilities of secretome as an alternative regenerative treatment other than cell-based regeneration treatment for the patient with coronary artery disease. In this early research we used limited patient but we will expand further the research with involving more samples and aim to have clinical trials should we achieve satisfying results. We also consider to combine the secretome treatment with existing coronary artery disease treatment, to ensure the usage of secretome combined with current medical treatment will benefit synergistically. Q: Other statins should also be investigated as they are being used widely in real world. A: Thank you for the feedback, we will consider that as our future research suggestion. However, we first prefer atorvastatin since it is readily available in our country, Indonesia. Previously, we have compared the effect of atorvastatin, rosuvastatin and simvastatin effect on the EPCs (without secretome) and conclude that atorvastatin was the most superior in inducing EPCs migration. Thus, this research prefers to use atorvastatin and combine it with secretome. Q: The exact composition of the hUCB-MSc derived secretome should be described. A: Thanks, we would like to admit that the limitation of this research was the inability to exactly describe the molecule in the hUCB-MSc derived secretome. Further research is required through proteomic analysis to determine the molecule in the hUCB-MSc derived secretome. This will also help to standardize the composition of hUCB-MSc derived secretome. Again, many thanks for the review,, Best Regards Yudi Her" } ] } ]	1	https://f1000research.com/articles/9-537
https://f1000research.com/articles/10-121/v1	17 Feb 21	{ "type": "Study Protocol", "title": "Modular intervention to improve paternal involvement and support for better infant and young child feeding in a district of coastal South India: a randomized controlled trial protocol", "authors": [ "Prasanna Mithra", "Bhaskaran Unnikrishnan", "Rekha Thapar", "Nithin Kumar", "Ramesh Holla", "Priya Rathi", "Bhaskaran Unnikrishnan", "Rekha Thapar", "Nithin Kumar", "Ramesh Holla", "Priya Rathi" ], "abstract": "Background: The major determinant to the well-being of infants and young children (IYC) is their feeding practices. These practices are the responsibility of both parents, meaning that fathers have an equal role to mothers. Fathers’ involvement can have an impact on the overall health of the children. Despite this, paternal involvement towards IYC feeding (IYCF) have not been studied adequately. Methods: This randomized control trial (n=120) will be conducted among fathers of infants (children aged <1 year) and young children (children aged 12-23 months) in selected households in Dakshina Kannada District of the southern Indian State of Karnataka. The study will be conducted after an initial baseline assessment on awareness, attitude and involvement of fathers in IYCF. Fathers with scores less than the 50th percentile in the practice component will be categorized as fathers with poor involvement and will be potential participants for the trial. A visual module will be developed and validated for improving paternal involvement in IYCF. Using a simple randomization technique, the participants will be allocated to modular intervention and control group (1:1 allocation). Each participant in the intervention arm will be visited once a month to implement the module, for six months on a one-to-one basis. Following the intervention, a post-test assessment will be done for both groups to measure the level of paternal involvement in IYCF. Ethics and dissemination: Approval has been obtained from the Institutional Ethics Committee of Kasturba Medical College, Mangalore, India. The dissemination plans include scientific conferences and publication in scientific journals. Registration: The study is registered with Clinical Trial Registry of India (CTRI/2017/06/008936).", "keywords": [ "IYCF", "father", "involvement", "module", "child", "development" ], "content": "Introduction\n\nFeeding practices are one of the major determinants of the survival and wellbeing of children1. Providing good nutrition to a child is the responsibility of both parents. Fathers are proficient caregivers; their positive involvement along with mothers in child feeding and rearing is associated with overall health outcomes2.\n\nThe currently available literature points towards the good impact of fathers’ involvement in infants and young children feeding (IYCF) and rearing on the health of their children3. Also, the manner in which fathers assist and support their spouses in terms of child nutrition determines long-term effects4. It has been documented that such positive behaviour from fathers results in a lower BMI among adolescent girls4. Several strategies have been tried to enhance IYCF, including breastfeeding, such as establishing standards in providing maternity services through building public awareness using mass media, peer-support interventions, and health care provider driven initiatives providing maternal support5. Observational studies have documented that the early provision of support from fathers and their active participation in breastfeeding practices, which could happen with adequate knowledge and a positive attitude, had effects on the initiation and overall duration of breastfeeding5.\n\nThere are many socio-demographic factors playing an important role in paternal involvement in infant feeding practices, but there is lack of evidence to suggest the extent and determining level of paternal involvement6–8. Also, the module-based interventions for fathers to enhancing their involvement in IYCF have not been tested.\n\nThere are not many documented studies from India in this regard. This study would provide insight about the above stated priority areas and help investigators to develop strategies to enhance infant and young child nutrition, provide a modular intervention to fathers on IYCF, and then evaluate the intervention.\n\nFathers play an important supporting role in overall maternal and child health9 and their views of child-feeding practices influence development of the child and eating behaviour10–12. As a result of changes in societal norms, fathers’ involvement in childcare has increased compared to the level a few years ago. Increased involvement is influenced by several sociocultural, behavioral, hormonal, and neural factors13. With the birth of a first child, a man enters the stage of fatherhood; which is a crucial and challenging period14. If this entry to fatherhood becomes smoother, then the likelihood that the father being more involved in childcare is higher. This can happen with adequate knowledge transfer to the fathers of young children and prospective fathers13. However, there are limited studies that have been done among fathers regarding their involvement in IYCF11. In the Indian scenario, the evidence related to paternal involvement and measures for its enhancement is scant. The focus of interventions and studies related to child feeding are mostly on mothers, since mothers spend the most amount of time with children than anyone else in the family10.\n\nAcross different regions of the world, studies done on fathers of young children were mostly of exploratory nature to assess the factors determining IYCF practices, qualitative assessments, couple-oriented interventions, family-based interventions, etc. This limited involvement from the fathers’ side were influenced by several region- and culture-specific challenges they faced3,9,15,16. However, in countries like the UK, it was made a strategic priority to enhance breast feeding practices and it was realized that this could be achieved with a “strong social support from their partner”17. It was also felt that fathers need informed guidance in supporting their spouses on breastfeeding and IYCF17. There is no consensus on measuring male involvement and few beliefs exist that imply male involvement could be a threat to women’s empowerment and autonomy in terms of child rearing practices18. Across the studies, there were felt needs from mothers and fathers of the young children regarding increasing the paternal involvement in the child feeding and care. Behaviour Change Communication strategies have been frequently used among mothers. Such strategies, when centered around both the parents have a higher effect on the father’s knowledge and involvement in child feeding, as compared to targeting mothers only. An increase in knowledge, however, may not have the desired impact on IYCF practices19.\n\nMaycock et al. carried out a randomized control trial (RCT) in Perth, Western Australia, known as the Fathers Infant Feeding Initiative (FIFI Study). The trial evaluated the effect of an antenatal paternal education session and spousal support during the postnatal period, targeted at fathers during the antenatal and postnatal period of their spouses. The study reported a significant increase in breastfeeding rate at 6 weeks. They also reported a correlation between a higher age of fathers and a high socio economic status with higher rates of breastfeeding at six weeks20. Abdullahi et al., in Somalia, reported a quasi-experimental study in 2019, commissioned by Save the Children International (SCI), aimed at assessing the effects of peer counselling by Mother-to-Mother (M2M) and Father-to-Father (F2F) support groups on IYCF practices. They observed that there was IYCF knowledge growth, improvement in practices of breastfeeding and diet diversity among intervention arms. Since the follow up duration was short, there was no noticeable change in the nutritional status of children21.\n\nIn Japan, Ito et al. reported an inverse relation between infant care by fathers and breastfeeding during the early infancy (first 6 months of life); fathers’ likelihood of involvement was higher with higher education status, being unemployed and nonsmoking3. Pisacane et al., in Naples, Italy, investigated the effect of providing support to fathers in identifying their role in IYCF and teaching them to take care of most common lactation problems. They found that this intervention would result in higher rates of full breastfeeding at 6 months5. Sherriff et al., in Southern England, studied the main attributes of father support in relation to breast feeding. They identified knowledge, attitude and involvement in decision-making as key factors towards successful breastfeeding practice22.\n\nIn rural Kenya, the quasi-experimental study with pre- and post-test observations conducted by Thuita et al., studied the impact of engaging fathers or grandmothers in improving diets of mothers and IYCF feeding practices through “parallel peer education dialogue groups with fathers and grandmothers.” They reported success in bringing increased paternal engagement in IYCF23.\n\nOne study conducted among 210 father-young child dyads in Bangalore, India, in 2020, by Inbaraj et al., aimed to “explore paternal child-feeding patterns, their involvement in feeding, and its association with level of malnutrition in the slums”. The overall involvement in child feeding and rearing was shown by nearly half of the study participants. Religion, family types, and individual income were factors associated with poor involvement of fathers in child feeding10.\n\nIn 2014, Khandpur et al. reviewed the existing evidence on child feeding research in 20 studies, which included fathers’ feeding practices and their characteristics and correlations. They included studies which reported the feeding practices of fathers and/or primary care givers towards children aged up to 18 years. Most of the studies reported self-reported child feeding practices without a specific paternal validation. There were also studies reporting fathers pressuring their children to eat. Paternal and maternal feeding practices varied, wherein fathers monitored less and put more limitation to access to child’s food in comparison to mothers. Body fat of children was associated with fathers’ feeding practices11. This review also brought out the paucity of literature on fathers’ child feeding practices.\n\nTo assess the effectiveness of a module-based intervention for improving the paternal involvement in IYCF.\n\n1. To design and develop an educational module for the fathers on improving father’s IYCF practices and support.\n\n2. To evaluate the effectiveness of module in improving the level of paternal involvement in IYCF\n\n\nMethods\n\nVersion: 1.0 (22-12-2020).\n\nThe study area will be in the Dakshina Kannada District in the southern part of Karnataka State in South India. Regarding health care facilities, it is a progressing area. According to the 2011 Census report, the population of this area was 2,083,625, with a literacy rate of 88.62%24. The current study is being done by the Department of Community Medicine, Kasturba Medical College, Mangalore, Manipal Academy of Higher Education, Manipal, India.\n\nCommunity healthcare settings in Dakshina Kannada District, Karnataka State, India.\n\nThe study will be a unblinded parallel group RCT. This study will follow the Consolidated Standards of Reporting Trials (CONSORT)25,26. The study flow according to CONSORT guidelines is depicted in the Extended data27. This study protocol is reported along with Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) Guidelines28. A completed SPIRIT checklist can be found in the Reporting guidelines27.\n\nFathers attending to infants (children aged <1 year) and young children (children aged 12–23 months) in selected households belonging to the geographical areas covered by public health care institutions of Dakshina Kannada District will be included. Households will be identified through records maintained in at public health care institutions. At the time of visit to their houses, their identity will be verified, along with confirmation of the age of their child.\n\nWith anticipated improvement in the paternal involvement of IYCF following the modular intervention as 15%, 80% power, 95% Confidence Interval, 1:1 allocation and along with addition of 20% non-response error, the total sample size will be 60 subjects.\n\nThe study will be carried out for a total period of one year (between January 2020 and December 2020).\n\nFathers of infants and young children with poor involvement in IYCF in households in Dakshina Kannada District will be selected for this study. Initial assessments of all fathers from selected households (see Study population) will be conducted to assess fathers’ knowledge, attitude and practices towards IYCF (see questionnaire in Extended data27). Fathers with scores in the lower 50th percentile in the practice domain will be categorized as fathers with poor involvement in IYCF and will be eligible for inclusion in the RCT.\n\nFathers who score higher than the 50th percentile will be excluded from taking part in the RCT. In addition, fathers who are not available for interventions despite three attempts of contact will be excluded.\n\nTo promote retention of the participants, visits will be made to them at a convenient time and location. Intention to treat analysis will be performed (as mentioned in Data analysis).\n\nBased on the findings from initial assessment at the district for level of knowledge, attitude and practices towards paternal involvement in IYCF, the areas with fathers of infants with poor IYCF practices will be mapped for visits at their households. Selected eligible participants will be allocated to modular intervention and control groups. A simple randomization technique with 1:1 allocation will follow. A lottery technique will be used for randomization, until the needed sample size is reached, with the help of Microsoft Excel software. The principal investigator will generate the sequence for randomization and another author (NK) will enroll the participants according to this sequence. The research assistants will implement the modular intervention.\n\nAn IYCF improvement module that has been developed as an easy to use flip chart with a blend of pictures and textual materials has been produced (IYCF module, Extended data:27), which included the basic aspects of infant and child nutrition and feeding: (i) assisting in feeding; (ii) changing diapers; (iii) giving a bath to the child; (iv) putting the child to sleep; (v) playing with the child and (vi) taking the child outside the home, (e.g. - for a walk).\n\nThe module was developed based on the available literature and brainstorming sessions with the researchers. The module will be implemented in the local language (Kannada).\n\nThis module will be administered to the intervention group only. The control group will receive regular care at the study centers, which is routinely made available to them whenever they visit health centres or when health workers visit them. This will not affect the ultimate results of the study. The administration of the module to the intervention group will be done monthly for six months. Both groups will be followed up at the end of six months, during which they will be assessed for the level of involvement in IYCF, which is the same method as that of pre-assessment (see questionnaire, Extended data27).\n\nPre-tested, validated, semi-structured proforma will be used both pre- and post-intervention (questionnaire, Extended data27). The instrument will include the demographic details of the participants and Likert scores in these domains: knowledge, attitude and practices (involvement) towards IYCF.\n\nNecessary permissions are taken from the Head of the Institute and District Health Authorities. Two Research Assistants will be recruited from the field of Social Work, with communication skills and ability to interact with the population using the local language. After their training on implementation of the module, they will visit the study participants for the implementation.\n\nThe fathers accompanying the infants will be approached and will be provided with the participant information sheet (Extended data27). Written informed consent will be taken from each of them by the research assistants and they will be allocated to one of the intervention groups and one-to-one interaction for module implementation will be followed as stated above. The assessment at six months will be done by the research assistants for both the groups with respect to knowledge, attitude and involvement (practices) in their IYCF.\n\nAll the data collected in the field will be managed at the central coordinating site, independent of funding organization. A separate Data Monitoring Committee (DMC) will not be constituted. The filled proforma will be edited for inadvertently missing information, related to the demographic component and the participants will be contacted and for missing details. The variables will be coded and entered in IBM SPSS Statistics for Windows (Version 25.0. Armonk, NY: IBM Corp). From this database, 10% of the data will be randomly chosen to validate against the proforma. If the error rate is >0.3%, then the data re-entry will be performed. The data forms will be accessible only to the study staff. The entered data will be secured with a password protected access. The monitoring of data will be undertaken by the administrative staff of the study department and they are not part of the study.\n\nResults will be expressed as proportions, mean (with standard deviation), median (with interquartile range), using appropriate tables and figures. We will follow intention to treat analysis (ITT). The comparison for the continuous variables will be done using the “t” tests. For comparison across the groups, Chi square tests and Multiple Logistic Regression will be used and p<0.05 will be considered statistically significant.\n\nThe level of involvement of fathers in IYCF, which is of paramount importance to the overall nutrition of children, and extent to which the modules will achieve improvement. In addition, change in knowledge and attitude towards IYCF will be assessed at the end of six months of intervention. The outcomes assessed will be knowledge scores, attitude scores and practice (involvement) scores obtained through the proforma.\n\nThe current research findings would further be emulated in other regions of the country and elsewhere. Also, the long-term impact of the modular interventions would attract future studies. This in turn would benefit the practitioners and policy makers in enhancing the paternal involvement in IYCF and planning the interventions accordingly.\n\nThe study protocol has been approved by the Institutional Ethics Committee (IEC) of Kasturba Medical College, Mangalore, Manipal Academy of Higher Education, Manipal, India. Participants will be given an information letter, and written informed consent will be taken from each of them. All the information collected will be kept confidential. The study has been registered with the Clinical Trial Registry of India (CTRI/2017/06/008936)29.\n\nThe findings of this study will be disseminated through presentations in scientific conferences and scientific journals. Brief policy notes will be prepared and shared with local health authorities.\n\nWe will use the CONSORT guidelines, as mentioned in Study design, to report the study flow and findings26,30. For describing the methodology of this trial, we will use the TIDieR Checklist31.\n\nThe IEC guidelines of study institute do not permit sharing of research data with any external agency, because they contain personal information and could lead to breach of confidentiality, assured to the participants. However, on personal request and appropriate justification, the data can be obtained from the corresponding author.\n\n\nCurrent status of the study\n\nThe study has completed recruiting the participants and four rounds of monthly modular interventions have been administered.\n\n\nData availability\n\nNo data is associated with this article.\n\nOpen Science Framework: Modular intervention to improve paternal involvement and support for better infant and young child feeding (IYCF) in a district of coastal South India - A randomized controlled trial (Protocol), https://doi.org/10.17605/OSF.IO/4UB7G27.\n\nThis project contains the following extended data:\n\n- Informed consent form and information sheet\n\n- Questionnaire\n\n- IYCF module\n\nOpen Scientific Framework: SPIRIT checklist and CONSORT flowchart for ‘Modular intervention to improve paternal involvement and support for better infant and young child feeding (IYCF) in a district of coastal South India - A randomized controlled trial (Protocol)’, https://doi.org/10.17605/OSF.IO/4UB7G27.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nWe acknowledge Kasturba Medical College, Mangalore, Manipal Academy of Higher Education, Manipal, India and Indian Institute of Public Health, Gandhinagar, India for the support rendered to carry out this study.\n\n\nReferences\n\nRao S, Swathi PM, Unnikrishnan B, et al.: Study of complementary feeding practices among mothers of children aged six months to two years - a study from coastal south India. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTohotoa J, Maycock B, Hauck Y, et al.: Supporting mothers to breastfeed: The development and process evaluation of a father inclusive perinatal education support program in Perth, Western Australia. Health Promot Int. Oxford Academic; 2011; 26(3): 351–61. PubMed Abstract \| Publisher Full Text\n\nSloand E, Gebrian B: Fathers clubs to improve child health in rural Haiti. Public Health Nurs. 2006; 23(1): 46–51. PubMed Abstract \| Publisher Full Text\n\nYogman M, Garfield CF: Fathers’ roles in the care and development of their children: The role of pediatricians. Pediatrics. American Academy of Pediatrics; 2016; 138(1): e20161128. PubMed Abstract \| Publisher Full Text\n\nBrown A, Davies R: Fathers’ experiences of supporting breastfeeding: Challenges for breastfeeding promotion and education. Matern Child Nutr. 2014; 10(4): 510–26. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDumbaugh M, Tawiah-Agyemang C, Manu A, et al.: Perceptions of, attitudes towards and barriers to male involvement in newborn care in rural Ghana, West Africa: A qualitative analysis. BMC Pregnancy Childbirth. 2014; 14(1): 269. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHan YE, Park S, Kim JE, et al.: Father Engagement in Improving Infant and Young Child Feeding (IYCF) Practices: Evidence from a Clustered Randomized Controlled Trial in Ethiopia (P11-112-19). Curr Dev Nutr. 2019; 3(Supplement_ 1). Publisher Full Text\n\nMaycock B, Binns CW, Dhaliwal S, et al.: Education and support for fathers improves breastfeeding rates: A randomized controlled trial. J Hum Lact. 2013; 29(4): 484–90. PubMed Abstract \| Publisher Full Text\n\nAbdullahi LH, Sheikh A, Elijah M, et al.: Save the Children Somalia Enhancing Infant and Young Child Feeding Practices in Somalia: Effect of Peer Counselling through Mother-to-Mother and Father-to-Father Support Groups. 2019. Reference Source\n\nSherriff N, Hall V, Panton C: Engaging and supporting fathers to promote breast feeding: A concept analysis. Midwifery. 2014; 30(6): 667–77. PubMed Abstract \| Publisher Full Text\n\nThuita FM, Martin SL, Ndegwa K, et al.: Engaging fathers and grandmothers to improve maternal and child dietary practices: planning a community-based study in western Kenya. African J Food Agric Nutr Dev. 2015; 15(5): 10386–10405. Reference Source\n\nStatistical Report \| District Dakshina Kannada, Government of Karnataka \| India. [cited 2020 Nov 13]. Reference Source\n\nMoher D, Schulz KF, Altman DG: The CONSORT statement: revised recommendations for improving the quality of reports of parallel-group randomized trials. J Am Podiatr Med Assoc. 2001; 91(8): 437–42. PubMed Abstract \| Publisher Full Text\n\nConsort: The CONSORT Flow Diagram. [cited 2020 Nov 13]. Reference Source\n\nMithra P, Unnikrishnan B, Rekha T, et al.: Modular intervention to improve paternal involvement and support for better infant and young child feeding (IYCF) in a district of coastal South India - A randomized controlled trial (Protocol). 2021. http://www.doi.org/10.17605/OSF.IO/4UB7G\n\nThe SPIRIT Statement. [cited 2020 Dec 22]. Reference Source\n\nCTRI. [cited 2020 Dec 22]. Reference Source\n\nMoher D, Schulz KF, Altman DG, et al.: The CONSORT statement: Revised recommendations for improving the quality of reports of parallel-group randomised trials. Lancet. Elsevier Limited; 2001; 357(9263): 1191–4. PubMed Abstract \| Publisher Full Text\n\nHoffmann TC, Glasziou PP, Boutron I, et al.: Better Reporting of Interventions: Template for Intervention Description and Replication (TIDieR) Checklist and Guide. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "79720", "date": "04 Mar 2021", "name": "Sudhir Prabhu H", "expertise": [ "Reviewer Expertise Public health", "Community Medicine", "Child health and immunization" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study is designed to address an important research question considering the societal context of the current paternal role and means to strengthen it for infant care and feeding through a modular intervention targetting fathers. The authors have done an extensive review on the topic and have designed an extremely sound study protocol to ensure generation of good scientific data for policy and program on child care.\nHowever, elaborating or modifying a few of the points below may make the protocol more robust prior to begining data collection:\n1. Methods section -\nStudy population is being mentioned to be taken from households covered under public health care institutions of Dakshina kannada district. Sampling technique and enrollment is not being mentioned. It is not clear how many such institutions would constitute the primary sampling unit, will there be equal representation of locality (urban,rural) partners of working mothers, socio-demographic and culturally diverse population especially socially deprived communities being enrolled and how would researchers ensure data enrollment complete. Is it conveneient sampling till sample size is achieved?\n\nStudy duration is being mentioned as January 2020 to December 2020 and data collection methodology mentions permission from District health authorities. If data collection has not started it would be better to consider seeking permission from Indian council of medical research as per the circular sent by Directorate of Health and welfare services, Karnataka number NVBDCP/ENT04/2019-20 dated 8/4/2019 mentioning that only ICMR is competent authority to approve community based interventions for private medical college research projects in community area and not district health officer of the district.\n\nEligibility criteria: fathers scoring less than 50th percentile will be implied exclusion need not be stated again.\n\nIntention to treat analysis can be removed from methods section.\n2. Intervention procedure -\nModule development technique is not clearly stated - was it through Delphi or any other specific methods and who were the researchers who were involved? Module has images from google images and shutterstock.com which may be under copyright or royalty free clause. Kindly verify due permissions and regulations for the use.\n\nEven content of the module may need elaboration on specific paternal role for example sleep component mentions only sleep time and images which indicate baby being sleepy, the role of father getting involved is not stated explicitly similarly immunization and growth monitoring doesnt include Thaayi card (mother and child protection card) or immunization card which should be known to father as a go to chart when it comes to vaccination and growth and development details as well as picking up warning signs for poor milestones.\n3. CONSORT flow diagram mentioned can be more precise for exclusion, intervention and final outcome process.\n4. Study questionnaire has a mix of Likert, open ended and closed ended questions. If self-administered it may need to be told in detail to study participants before filling up the responses.\n5. Study outcomes and implications - only fathers knowledge, involvement is being assessed at 6 months assessing the health status, growth and development milestones of infant might be better outcome variables to look and understand when it comes to generating evidence.\n6. DK district having higher literacy and less Infant mortality and malnutrition cases among children study may have generalizability issues for the rest of the districts of Karnataka or India.\n7. Informed consent document and patient information sheet should have more details on what is a modular intervention, why and how were the study particpants selected and included in the final trial subject population, their expectations throughout the study period, details on outcome assessment should be clearly stated to study subjects.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "6641", "date": "10 May 2021", "name": "Prasanna Mithra", "role": "Author Response", "response": "Reviewer Comments; followed by the response (in bold) by Authors: The study is designed to address an important research question considering the societal context of the current paternal role and means to strengthen it for infant care and feeding through a modular intervention targetting fathers. The authors have done an extensive review on the topic and have designed an extremely sound study protocol to ensure generation of good scientific data for policy and program on child care. Thank you for the in depth review and constructive comments. However, elaborating or modifying a few of the points below may make the protocol more robust prior to begining data collection: 1. Methods section - Study population is being mentioned to be taken from households covered under public health care institutions of Dakshina kannada district. Sampling technique and enrollment is not being mentioned. It is not clear how many such institutions would constitute the primary sampling unit, will there be equal representation of locality (urban,rural) partners of working mothers, socio-demographic and culturally diverse population especially socially deprived communities being enrolled and how would researchers ensure data enrollment complete. Is it conveneient sampling till sample size is achieved? A new paragraph is now added in the “sampling strategy” section for enhanced clarity regarding the enrollment procedure. Study duration is being mentioned as January 2020 to December 2020 and data collection methodology mentions permission from District health authorities. If data collection has not started it would be better to consider seeking permission from Indian council of medical research as per the circular sent by Directorate of Health and welfare services, Karnataka number NVBDCP/ENT04/2019-20 dated 8/4/2019 mentioning that only ICMR is competent authority to approve community based interventions for private medical college research projects in community area and not district health officer of the district. At present the study stands completed. However, the permissions from the District Health Authorities were sought well before the enforcement of the above referred rule by Directorate of Health and welfare services, Karnataka State, India. Though, the study was carried out by a Private Institute, it had Department of Science and Technology (Govt of India) as the principal supporter through Indian Institute of Public Health Gandhinagar (Government Institute); which was also the technical partner. Eligibility criteria: fathers scoring less than 50th percentile will be implied exclusion need not be stated again. Thank you. The duplicate statement is now removed. Intention to treat analysis can be removed from methods section. Thank you. The repetition is removed and the mention of intention to treat analysis (ITT) in the methods section is deleted. The statement about ITT is retained only in “Data analysis” section. 2. Intervention procedure - Module development technique is not clearly stated - was it through Delphi or any other specific methods and who were the researchers who were involved? Module has images from google images and shutterstock.com which may be under copyright or royalty free clause. Kindly verify due permissions and regulations for the use. Technique used for the development of the module was “brainstorming technique”; which is mentioned as “The module was developed based on the available literature and brainstorming sessions with the researchers.” in the “Intervention procedure” section. To enhance clarity, a sentence is added about the details of investigators involved in developing the module. Regarding the images, google images used did not have copyright issues; however, we have mentioned the source of images at relevant locations. For “shutterstock.com” images, we had trial account subscription which would give 10 images free for the use. Even content of the module may need elaboration on specific paternal role for example sleep component mentions only sleep time and images which indicate baby being sleepy, the role of father getting involved is not stated explicitly similarly immunization and growth monitoring doesnt include Thaayi card (mother and child protection card) or immunization card which should be known to father as a go to chart when it comes to vaccination and growth and development details as well as picking up warning signs for poor milestones. The research assistants were trained to elaborate and explain these specific role of fathers in putting the baby to sleep and in following up on baby’s immunization. Due to several variety of maternal and child protection cards which are commonly used by the families in this area, they were not included in the module as image except for the standard growth chart. However, these cards were scrutinized by the research assistants in the field. 3. CONSORT flow diagram mentioned can be more precise for exclusion, intervention and final outcome process. Thank you. CONSORT flow diagram is now made more precise with respect to these characteristics. 4. Study questionnaire has a mix of Likert, open ended and closed ended questions. If self-administered it may need to be told in detail to study participants before filling up the responses. The data collection tool is research assistant administered. Hence the study participant would not go through the challenge of filing variety of questions. 5. Study outcomes and implications - only fathers knowledge, involvement is being assessed at 6 months assessing the health status, growth and development milestones of infant might be better outcome variables to look and understand when it comes to generating evidence. Thank you for the suggestions. However, the objective of the intervention is only to target the paternal knowledge, attitude and involvement (practice) towards IYCF. Assessing milestones and growth pattern would have demanded more work force; which was beyond the feasibility range of this interventional study. 6. DK district having higher literacy and less Infant mortality and malnutrition cases among children study may have generalizability issues for the rest of the districts of Karnataka or India. Thank you. This model of paternal intervention included participants from different strata of society and standard recruitment techniques were being followed to minimize the challenges of generalization. Success of this model could also be tested in other parts of the country or elsewhere. Moreover, this trial also provides a guide to feasibility aspects. 7. Informed consent document and patient information sheet should have more details on what is a modular intervention, why and how were the study particpants selected and included in the final trial subject population, their expectations throughout the study period, details on outcome assessment should be clearly stated to study subjects. The entire consenting process involved a detailed conversation between the research assistant in the field and the study participant. Therefore, the participant information sheet provided herewith is one part of the consenting process and the conversation uniformly involved explanations about the nature of intervention, frequency of contact, post intervention assessment and parameters which would assessed and measured." } ] } ]	1	https://f1000research.com/articles/10-121
https://f1000research.com/articles/9-1398/v1	04 Dec 20	{ "type": "Opinion Article", "title": "The need for standardisation in life science research - an approach to excellence and trust.", "authors": [ "Susanne Hollmann", "Andreas Kremer", "Špela Baebler", "Christophe Trefois", "Kristina Gruden", "Witold R. Rudnicki", "Weida Tong", "Aleksandra Gruca", "Erik Bongcam-Rudloff", "Chris T. Evelo", "Alina Nechyporenko", "Marcus Frohme", "David Šafránek", "Babette Regierer", "Domenica D'Elia", "Andreas Kremer", "Špela Baebler", "Christophe Trefois", "Kristina Gruden", "Witold R. Rudnicki", "Weida Tong", "Aleksandra Gruca", "Erik Bongcam-Rudloff", "Chris T. Evelo", "Alina Nechyporenko", "Marcus Frohme", "David Šafránek", "Babette Regierer", "Domenica D'Elia" ], "abstract": "Today, academic researchers benefit from the changes driven by digital technologies and the enormous growth of knowledge and data, on globalisation, enlargement of the scientific community, and the linkage between different scientific communities and the society. To fully benefit from this development, however, information needs to be shared openly and transparently. Digitalisation plays a major role here because it permeates all areas of business, science and society and is one of the key drivers for innovation and international cooperation. To address the resulting opportunities, the EU promotes the development and use of collaborative ways to produce and share knowledge and data as early as possible in the research process, but also to appropriately secure results with the European strategy for Open Science (OS). It is now widely recognised that making research results more accessible to all societal actors contributes to more effective and efficient science; it also serves as a boost for innovation in the public and private sectors. However for research data to be findable, accessible, interoperable and reusable the use of standards is essential. At the metadata level, considerable efforts in standardisation have already been made (e.g. Data Management Plan and FAIR Principle etc.), whereas in context with the raw data these fundamental efforts are still fragmented and in some cases completely missing. The CHARME consortium, funded by the European Cooperation in Science and Technology (COST) Agency, has identified needs and gaps in the field of standardisation in the life sciences and also discussed potential hurdles for implementation of standards in current practice. Here, the authors suggest four measures in response to current challenges to ensure a high quality of life science research data and their re-usability for research and innovation.", "keywords": [ "Open Data", "Open Access", "Open Science", "FAIR Principles", "Standardisation", "Education", "Quality Management" ], "content": "Introduction\n\nLife science research is the driver for biotechnological applications, one of the fastest-growing and potentially biggest future markets worldwide. As a result of advances in biological research and the support provided by information and communication technologies (ICT), new technologies have been developed that are changing our lives for example, by\n\nimproving healthcare (e.g., new prognostic and diagnostic tools, improved disease prevention strategies and treatments, nutritional health, exercise and risk evaluation, toxicology);\n\ncontributing to climate change mitigation and adaptation (e.g., by new fuels, new raw and renewable materials);\n\nincreasing the efficiency and sustainability of farming and food production (e.g. by improved crops, artificial meat, life stock, new fertilising and watering strategies as well as plant protection solutions);\n\nusing microorganisms as alternative production systems (e.g., using synthetic biology);\n\ndecreasing waste production (e.g., by the production of biodegradable materials);\n\nmodelling approaches leading to an increased understanding of how components in different systems interact and behave (e.g., by COMBINE, LiSyM);\n\nThese changes are directly connected to and driven by various factors such as globalisation, digitalisation and technology implementation. Digitalisation, in particular, plays a major role here because it permeates all areas of business, science, and society; and it is one of the key drivers for innovation and international cooperation. The European Union has generated initiatives to foster data openness and exchange, resulting in strategies like “Science 2.0”, “Open Research Data” and “Open Science”.\n\nIf these initiatives become successful by networking different ecosystems for the benefit of their participants, they will generate an immediate competitive advantage because research and innovation processes are increasingly taking place in cooperative value chains. Thus, it is important to emphasise that value chains cannot be developed in isolation, but are rather a global endeavour, and their success depends on open access to sources of a wide variety of knowledge.\n\nTo address the opportunities and challenges derived by new technological developments, governments and funding agencies promote the development and use of collaborative ways to produce and share knowledge and data as early as possible in the research process, but also to appropriately secure results. The EU has established the European Open Science Cloud as a constituting element that is intended to provide “.... access and reliable reuse of research data to European researchers, innovators, companies and citizens through a trusted [infrastructure]”. This strategy aims to enable science to become more efficient through better sharing of resources, more reliable through better validation procedures, and more responsive to society’s needs.\n\nTo facilitate research & innovation (R&I), one crucial step is to ensure high quality of the research data. High quality can only be achieved if data is generated according to well-defined and validated methodologies, using standards and proper means of quality control (QC). Most importantly, the use of standards and QC procedures must be applied consistently by the whole community for the data to be reproducible, easily shared and reused.\n\nThe “reproducibility crisis” within the life science research is a problem that emerged in recent years [citation in the note]. There are alarming reports from established representatives, like the Global Biological Standard Institute that in some fields such as cancer and preclinical research the majority of the published results are not reproducible1. The reasons for this “reproducibility crisis” are manifold, among them the lack of appropriate study design, proper controls, or insufficient documentation, etc. But the main reason is the absence of a unifying quality control and assurance framework. The ignorance of this aspect coms at high for the society, as recently stated “A 50% irreproducibility rate within preclinical research implies that more $28B/year is spent on research that is irreproducible”2. Properly established research standards the alignment of consensus-based best practices, reduce variance, and improve reproducibility and quality in research.\n\nA lack of interoperability is another factor that is currently often limiting the exchange and use of data from different sources. The need for data and tool interoperability and open standards to allow source connection was identified years ago. Still, only in recent years, the research community is pushing actors to develop proper tools and implement the use of standards. As summarised in an article of Sansone and Rocca-Serra in 2016, “...interoperability standards [should be recognised] as digital objects in their own right, with their associated research, development and educational activities”3. Full implementation of interoperability has not yet been achieved but needs to be fostered to tap the full potential of existing resources.\n\nIt could all be easy, but it is not. Detailed metadata descriptions and detailed annotations are required to enable and accelerate the desired innovation process. In life science research, there is a variety of languages, vocabularies, formats and methods that must be used, and not all necessary standards or tools that facilitate their automatic conversion are currently available. New technologies introduced in the last decade brought the flourishing of many new omics disciplines and multidisciplinary research approaches. This inherent heterogeneity of data sources and competencies now requires specific actions for the development and uniform adoption of standard procedures to be followed for enhanced reproducibility, sharing and reuse4. These actions are essential for advancing science and the utilization of research results for economic and societal benefits.\n\n\nThe CHARME community\n\nThe network of the COST Action CHARME (CA15110) is composed of a team of experts from different sectors of life science research and standardisation across 32 countries in Europe and beyond, has been working during 2016–2020 aiming to increase awareness for the need for standards, and identify critical existing gaps that need to be closed.\n\nAfter four years of successful work, the members of CHARME met in Brussels to summarise the achievements, to discuss future perspectives and challenges for standardisation in the life sciences and to elaborate a strategy to be suggested to the EC to accelerate the process of a wider application of standards in the life science research domain.\n\nFollowing the motto “Standards make the world go round”, the outcomes of the COST Action are manifold and introduced some basic concepts and definitions that support a better understanding of the challenges and requirements. However, these endeavours need to be accompanied by adequate policies. In the next section, we provide a list of challenges we have identified in the frame of standardisation that are key to improve the quality and innovation potential of the European life science research.\n\nThe first challenge identified is that the knowledge of standards and standardisation urgently need advertising and consequent awareness of the importance of their use. Many scientists are not aware of the standards in their field and suspect that the use of standards will limit their scientific freedom. It is exactly the opponents of standardisation that unconsciously use and appreciate these standards every day. Examples include operative systems like Android, formats like xml, pdf, jpg and QR- and barcodes, etc. It is essential for us to communicate the benefits of standards in the life sciences and the risks if standards are not acknowledged.\n\nThe second key aspect is to identify and fill evident gaps in education and training. Who knows how a standard is generated or what the difference between de facto and de jure standard is? It is of high importance to educate researchers that will become “next-generation” scientists being aware of quality management and standardisation in research. Besides, training and raising awareness is needed on all levels within the academic (and non-academic) system. Standards and quality management shake hands. A young researcher should get in contact with both issues as early as possible in the career, potentially already during his or her Bachelor studies. Learning about the basics like standard operating procedures (SOPs), quality control (QC) and data management should become as crucial as learning experimental design and how to document results.\n\nBecause the topic of implementation of standards is relatively new, and the awareness of its importance is rising in the scientific community, it is important to ensure proper training programmes for the university teachers. “Train-the-Trainer” programmes can multiply the effect to provide the necessary skills and knowledge to deliver courses on standards and quality management in research5.\n\nThe reproducibility crisis within life science research is an issue, that has emerged in recent years6. There were alarming reports from eminent representatives, such as the Global Biological Standard Institute that in some fields of the biological and clinical research, the majority of the published results are not reproducible. The cost for the society is high, as an example “A 50% irreproducibility rate within preclinical research implies that more $28B/year is spent on research that is irreproducible”2.\n\nThe reasons for this reproducibility crisis are manifold, among them the lack of appropriate study design, proper controls or insufficient documentation, etc. But the main reason is the absence of a unifying quality control and assurance framework. High-quality records are essential for the quality and reproducibility of research results and efficient technology transfer. Standards facilitate the alignment of consensus-based best practices, reduce variance, and improve reproducibility in research.\n\nAnother important issue is the verifiable origin of data. It is a crucial point for researchers but also for biotechnology companies.The documentation acts as a certificate for potential users (customers) and improves the traceability and transparency of the research process intending to prove the reliability of results. A good starting point for making a change is the introduction of quality documentation of experiments which is frequently an obvious lack within the process.\n\nFinally, to generate accurate and reproducible data sets for inter-laboratory comparisons, as well as further and future use of research data, it is mandatory to work in line with well-defined and validated methodologies, in compliance with standards and, where appropriate, according to good laboratory practices (GLP) and to data management plans (DMPs) that are produced at the planning stage of the research cycle7. Data management and stewardship are both concepts enforced by funding agencies in very recent years. Currently there is an overall lack of experts, and only a few universities in Europe have professional profiles and personnel that can cover the needs of their laboratories and computational teams with this specific expertise.\n\nAlong the pipeline of acquisition of data to (re)use we need to implement standards for description of samples and experiments, and standardised processes in the research workflows from the beginning.\n\nIn the context of data acquisition and storage, the potency for interoperability and transferability between different data formats and tools is likewise also limited. Whereas massive efforts have been made at the metadata level (data management plans, FAIR Data Principles, etc.), in context with the tools that can allow the easy and fast exchange of data amongst different data platforms or laboratories or between both, are still limited to a few research areas of life sciences.\n\nAn example is represented by electronic lab notebooks. They are relevant instruments to support the implementation of standards in the daily practice guiding the research process documentation. The connection of diverse ELN systems amongst each other, the study capture databases and the data repositories, and the standard development needed for that, were one of the main outcomes of CHARME. Nevertheless, this is what we see as one of the areas, if not a major area, where further adaptation of standards will help most to get real data to reuse.\n\nIt is important to understand that standardisation does not necessarily mean the refinement or development of standards. Conversely and hopefully, whenever possible, standardisation rather means applying existing standards, whether de facto or de jure8. The generation of standards in the sense of standardisation bodies like the International Organisation for Standardisation (ISO) or similar is a complex, time-consuming and cost-intensive process. In life science research and innovation processes, the development of an ISO standard often takes longer than the contract term of the project staff. Moreover, all the ISO standards should be reviewed every five years in terms of relevance to the marketplace.\n\nTo make matters worse, the use of standards typically requires the prior purchase of the document, making them very unattractive for most academic researchers. Another difficulty is the fact that there is currently no recognition for the contribution to the development of standards. Authors are not mentioned or acknowledged (i.e., the standards are created almost anonymously), and they do not count as a scientific track. Authorship recognition also affects the publication of raw data uploads to public repositories. Decision-makers, funding agencies and institutions urgently need to rethink if science should adopt and contribute to the overall process.\n\nIncentives should also be available for those academic or research institutions that profuse efforts toward the creation of virtuous research value-chains to make their research innovative and of high quality. This implies to invest in personnel and technologies for the development of projects and the production and management of data in line with FAIR principles and Open Access. Here we stress the needs and recommendations in the document “Policy Recommendations - Cost-Benefit analysis for FAIR research data”9. In this document, the section “Secure public funding for implementing and sustaining FAIR research data implementation” includes, among others, the need for FAIR-related costs, such as data stewardship and management, or data infrastructure operational costs to be made eligible for public funding. Specifically, Rec. 27 asks for: “research funding for FAIR data should continue being available not only at the European level, for example as part of Horizon 2020 and Horizon Europe, but also as part of national funded research and innovation programmes”.\n\nOn the overall, being the standards at the basis of FAIR data principle application, a specific focus on all the aspects related to the cost of their implementation also in terms of human resources should be taken under serious consideration.\n\nThe insufficient quality of the published data in the life-sciences tremendously reduces public investment in research. Current evaluation of the published data is leading to a worst-case scenario, e.g. for the medical sciences that up to 80% of the available data are not reusable. To avoid such waste, standards need to be developed and implemented for the entire research life cycle. Achieving a long-term and sustainable improvement requires not only awareness, engagement, training and education, but also an investment in infrastructure and personnel. Technical infrastructure needs to be established, maintained and constantly updated to allow an efficient and secure storing/hosting of the data generated by the scientific community. Public repositories have already been established offering the archiving of scientific data for all disciplines like OpenAIRE or specific research fields, like SEEK for the medical sciences. These repositories are only valuable if the data and associated metadata are of sufficient quality and following minimal standards. Therefore, also internal technical infrastructure hosted by research institutes should be established in a way that standards are applied. For providing technical solutions, but also for training, on-site advice and help, additional investments in personnel are required.\n\nConsidering that a broader implementation of standards within the scientific system might need long-term investments and commitment to introduce a change in how we perform science, it must also be considered what we put at risk:\n\n“Interpreting the overall cost of not having FAIR research data as a single value overlooks many non-quantifiable benefits of FAIR. Nonetheless, at €10.2bn per year in Europe, the measurable cost of not having FAIR research data makes an overwhelming case, in favour of the implementation of the FAIR principles”9.\n\nAlthough most stakeholders would agree that the way we perform research must change, the questions of who has the responsibility (and power) to introduce the change within the system and how we can finance an efficient support system are open to debate.\n\nThere is a need for European decision-makers to implement actions supporting all areas of the life science research (academic and non-academic) for equal opportunities and access to technology infrastructures, education programmes, and funding.\n\n\nCHARME outcomes and discussion\n\n“Making a step towards a European concept for excellence and trust in life science research”.\n\nImproving access to and management of data is fundamental, and we see that the importance of standardisation approaches for data collection, data description, application of the FAIR principles and for data modelling is increasingly recognised. But many of these efforts are still scattered, the awareness for available solutions is scarce, and there is missing recognition that standards still need to be harmonised, connected and further developed to make the system efficient. In general, this is about harmonising the field to maximise the way we benefit from already ongoing activities. That still needs better integration of ongoing initiatives on all the aspects mentioned.\n\nIn response to these challenges and to ensure the high quality of life science research data and their usability for R&I, measures must be taken at several levels to build an ecosystem of excellence and trust. Here we describe a series of measures that the CHARME network has identified as essential to this aim.\n\n\n\nConnection of existing and new local, national, European and international services into one interacting end-user service system, which should also strive for harmonisation with non-European efforts. That is, developing a structure in which communities jointly define standards according to standardisation bodies and guidelines from existing standardisation initiatives. This system should include the collection of information on standards and SOPs from different domains and enabling interaction with existing frameworks for agreement on nomenclature, ontologies and their international adoption. Integration and connection of existing expertise and services avoid heterogeneity of activities and ensures inclusiveness of all actors. Directly related to the need for connection and interaction between the existing facilities is the agreement about standard formats and operational procedures to enable reuse and reproducibility of data.\n\nExperts on this service structure should formulate advice to roadmapping efforts to indicate where standards need to be linked, identify overlaps and what kind of interoperability services still need to be developed. This structure should include a helpdesk and advice centre providing support and information advice that can point people to the relevant aspects but also a collection of standards and SOPs for different research domains. Several examples of suitable solutions for the use of SOPs in different life science domains already exist. For example, huge efforts have been made in the systems biology field for modelling and data exchange such as COPASI, CellNetAnalyzer, SABIO-RK and the SEEK platform of FAIRDOMHUB. The SEEK platform is a web-based resource for sharing diverse scientific research datasets, models or simulations, processes, protocols, SOPs and research outcomes. Another important initiative is EOSC life which is expected to act as an umbrella project for initiatives and projects like ELIXIR-Converge, the various life sciences related ESFRI and IMI projects and the GO-FAIR implementation network.\n\n\n\nThere are already many ongoing training initiatives (e.g., TeSS, the ELIXIR’s Training Portal, Train at EMBL-EBI, The Carpentry, GOBLET, FAIRsFAIR, etc.) and mostly every larger research project has its training work package or training module. Each of them provides training for their specific community. Although these training activities are highly relevant with an increased reach, they are not sufficient to address the entire research community and cover the specificities of the broad spectrum of the life science fields. To solve this, we need to join efforts and develop a common capacity building framework by coordinating training activities led by international, high quality, collaborative Science & Technology (S&T) networks. This will bridge and connect research disciplines also content-wise and link the standardisation approaches they use. This will also enable interdisciplinary cooperation by the integration of expertise from nearby research sectors/communities in life-science research (systems biology, systems medicine, medicine, biotechnology, plant science, computational biology and bioinformatics, etc.), and allow breakthroughs in scientific development built on synergistic efforts.\n\nMore specifically, the following measures must be addressed:\n\n• Concerted training actions facilitating the expansion of the currently sparse critical mass of specialists and experts who understand and possess knowledge of the principles and relevant tools within Europe. Cooperation of world-leading experts within each scientific field thereby will ensure mutual benefits and the best possible platform for any research and educational activities. Current collaborations among European and non-European organisations are e.g., the CABANA project, GOBLET-EMBnet and ISCB (North America), SoIBio (in South America), ApBioNet (Asia), ASBCB and H3ABioNet (Africa).\n\n• Train-the-Trainer. A major effort should be made for enlarging the offer of train-the-trainer programmes to allows trainers to acquire good training practices embracing standards5. Such action would enforce and multiply the spreading of knowledge in good practices for life science research and computational biology in the academic world and beyond.\n\n• Curricula. Besides offering training modules in undergraduate and/or graduate programmes, additional approaches might be worthwhile to consider engaging young scientists in the appreciation and use of standards. Students might be offered the possibility to participate in standards-related activities, e.g. in programming sessions. For example, COMBINE (“COmputational Modeling in BIology Network)” is an initiative to coordinate the development of the various community standards and formats for computational models. Alternatively, they could be involved in the development and testing of SOPs. Such activities should be linked to a credit point system incentivising their contribution. Benefits might be manifold: the students have the chance to learn hands-on with experts, they might even have the chance to be an author in a publication, the university would gain new knowledge and expertise, and the standardisation community is sustainably supported by engaged young researchers.\n\n• New professorships. Establishment of a research agenda in the field of standardisation and innovation transfer by (endowed) chairs on standardisation and innovation throughout Europe.\n\n• Train the experts. Training in standards, standardised processes and standardisation frameworks should also include training of established researchers and supervisors because they are often acting as reviewers and evaluators, and should be sensitised for the topic and appreciate the relevance.\n\nAny construct that provides activities as mentioned above in research, education, storing services and that is willing to interact and become a recognised member of the community should be exclusively designated following the measure pointed out in Measure 3.\n\n\n\nWe think that there is a strong need for mechanisms of control for the quality of openly accessible data. This data check must be upstream of the open access. A “seal of quality” similar to a DMP with a clear definition of quality benchmarks for data is needed in order to define metrics which are applicable and reasonable for building a framework around good data quality10 which are unthinkingly usable for further proceeding by everyone. This seal of quality should be supported by incentives from funders and publishers. Incentives should also consider another important aspect, that is education to acceptance of quality assurance (QA) plans. There are many examples of resistance of researchers to accept rules that QA plans impose and how these resistances can be easily overcome by education11.To enlarge as much as possible the possibility for courses and implementation of these courses as part of university curricula is a crucial step forward the suniversalisation of a safe and reliable way to do research.\n\n• Introduction of measures for QMS in funding programs. QMS will ensure quality assurance and reproducibility of research data which are prerequisites for all knowledge transfer activities in life-science research, and thus will have a substantial impact on technology transfer. This will contribute to increase and foster interdisciplinary and transnational collaboration between stakeholders from academia and industry and create a network with strong potential to impact and address social, ecological, economic challenges. The introduction of QMS should be done in a top-down approach like the implementation of DMPs, at a first stage voluntarily and connected to incentives and recognition for scientists who follow the principles.\n\n• Awarding a “Seal of quality” by the development and application of a review mechanism system that selects, appraises and monitors the performance of institutions, centres, platforms, or infrastructures. Such an accreditation-like system would ensure that high-quality performance of institutions in their implementation of internal policy and provision of infrastructure for data management and standards are visible and acknowledged.\n\nA best practice model for such a system is the Organisation of the European Cancer Research Institutes (OECI), who developed a catalogue of criteria that ensures high quality research from the participating institutions. Now all cancer research institutes and university clinics need to have at least a minimum of criteria to be fulfilled to be part of the OECI. This seal of quality could not only become an indicator for the researcher’s selection and decision for future employers but may also become a criterion for faster acceptance of publications. The same rating approach should be applied to datasets. Datasets frequently used by the research community and found to be reliable in terms of their FAIRness and reproducibility should get high ratings. Researchers should be able to share their experience when they use a particular dataset. This system would also be transferable to computational models to ensure reproducibility of the modelling based on the model parameters given and the dataset provided. For example, the MAQC society is now focused on establishing evaluation systems for the reproducibility of computer models based on first transcriptomics data. In future, they will extend these efforts to other omics data as well.\n\n\n\nStandards are important aspects in industry and ISO was founded with the idea of answering a fundamental question: “what’s the best way of doing this?”. Standards in life sciences are not only supporting knowledge and data exchange, but they are also ensuring the reliability of the materials, products, methods, or services and thus help to ensure product functionality, and support consumer safety and public health.\n\nCo-creation processes in standardisation are of mutual benefit and support knowledge and technology transfer, open innovation and cross-industry innovation. Although many standards have been developed in recent years, the insufficient implementation of and compliance to existing standards, e.g. within the life science sector, lead to a disruption of the innovation pipeline - often happening at the interface between academic research and industry - simply because of bad quality and missing reproducibility/reusability of the data.\n\nAcademics are often not aware of the necessity of standardised processes, especially in application-oriented work, which negatively affects or prevents academia-industry cooperation. In the development of drugs or new diagnostic tools, this ignorance to apply standards and quality management strategies leads to unnecessary disruption in the process because many results cannot be reproduced.\n\nThus, to facilitate the open innovation processes and to improve the collaboration between groups in academia and industry, partners from all sectors and stakeholder groups need to be involved as much as possible in standardisation activities launched by standardisation bodies. Still, also their voice needs to be considered in academia-driven grassroot standardisation initiatives.\n\nEducation in the field of standardisation at universities in the life sciences, management sciences, economics and law, therefore, is a key strategy to enable both, successful academia-industry cooperation and generation of innovation. A good example here is the Master’s course “Technopreneurship” (MTECH) at the University of Luxembourg aiming at the education of students to transfer smart secure ICT knowledge directly into technical innovation, through the prism of the competitive and innovative tool of technical standardisation.\n\nFinally, appropriate education in the context of standardisation leads to another advantage: skills in standardisation processes increase job opportunities in the non-academic sector. As mentioned already, knowledge about regulations and standards is crucial components to ensure best practice and help to ensure product quality and consumer safety. Therefore, it seems beneficial to:\n\ninvolve non-academic partners in the development of curricula at the undergraduate and graduate levels based on market needs;\n\nimplement mandatory seminars, colloquia provided by non-academic partners as well as study visits and traineeships in the industry into the curricula and the examination infrastructure;\n\nestablish standardisation and innovation transfer chairs.\n\n\n\nBased on the experience gained within the COST Action CHARME, we suggest the establishment of an umbrella hub (helpdesk) on standardisation. Similar to the IP Helpdesk of the European Commission, this construct will bundle and harmonise all initiatives and institutions that are already focussing on this topic and will represent a central hub for all activities related to standards and standardisation.\n\nThis hub should:\n\n1. Consist of experts from the different fields, institutions, bodies and initiatives and bodies in life science research mentioned above, and offer services to answer all the questions related to SOPs, protocols and data standards.\n\n2. Provide Open Access protocols, methods and tools serving as a basis for proper data management (SOPs, Standards) by establishing an Application Program Interface (API) to a queryable and searchable database of existing standards, SOPs, protocols and tools with links to the corresponding guardians, and actors and mechanisms for collaborative preparation.\n\n3. Adopt EU- wide training by providing Train-the-Trainer modules and sharing of high-quality teaching materials and methods among education professionals across borders.\n\n4. Include budget for funding programmes or projects, e.g. for the generation of tools enabling (cross-sectoral) interoperability and training.\n\n5. Become the contact point for the communication and collection of needs from the operational level, but also for translation and exchange with decision-makers.\n\n6. Be open to the public and free of charges, centralised and monitored by the EC.\n\n7. Become the core of a worldwide infrastructure and thus function as an integrators between grassroot scientific initiatives and activities governed by standardisation bodies.\n\n\nConclusions\n\nWe believe that this White Paper demonstrates the global need to promote standards in the life sciences research in response to a major challenge of implementing open science principles in the academic workflow, especially with respect to the reproducibility and reliability of research data. To promote these standards, we have identified four measures to support their implementation across the entire research community. Interactions with platforms and communities such as ELIXIR and worldwide integration in the complex landscape of societies, initiatives and projects have been addressed to avoid duplication of efforts and ensure fruitful collaborations. Finally, the growing interest for reproducible data will ensure the global recognition and expansion of the research community and will trigger numerous novel interactions between academia and industry. Europe is well-positioned for global leadership in building alliances around common values, and promoting FAIR principles and standardisation processes and developing a landscape of interoperability services to facilitate these connections.\n\n\nData availability\n\nNo data is associated with this article.\n\n\nDisclaimer\n\nW.T.: The views presented in this article do not necessarily reflect current or future opinion or policy of the US Food and Drug Administration. Any mention of commercial products is for clarification and not intended as an endorsement.", "appendix": "Acknowledgements\n\nWe thank all members of the Cost Actions Network CHARME for their contribution and support. We thank Dr Karl Grun, head of innovation in Austrian Standards, and S. Nik from CEN-CENELEC for their comments and the fruitful discussion of the paper’s content.\n\n\nReferences\n\nFreedman LP, Inglese J: The increasing urgency for standards in basic biologic research. Cancer Res. 2014; 74(15): 4024–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFreedman LP, Cockburn IM, Simcoe TS: The Economics of Reproducibility in Preclinical Research. [published correction appears in PLoS Biol. 2018 Apr 10;16(4):e1002626]. PLoS Biol. 2015; 13(6): e1002165. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSansone SA, Rocca-Serra P: Interoperability Standards - Digital Objects in Their Own Right. Wellcome Trust. 2016. Publisher Full Text\n\nHackett RA, Belitz MW, Gilbert EE, et al.: A data management workflow of biodiversity data from the field to data users. Appl Plant Sci. 2019; 7(12): e11310. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVia A, Attwood TK, Fernandes PL, et al.: A new pan-European Train-the-Trainer programme for bioinformatics: pilot results on feasibility, utility and sustainability of learning. Brief Bioinform. 2019; 20(2): 405–415. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBaker M: 1,500 scientists lift the lid on reproducibility. Nature. 2016; 533(7604): 452–4. PubMed Abstract \| Publisher Full Text\n\nWilkinson MD, Dumontier M, Aalbersberg IJJ, et al.: The FAIR Guiding Principles for scientific data management and stewardship. [published correction appears in Sci Data. 2019 Mar 19;6(1):6]. Sci Data. 2016; 3: 160018. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWikipedia contributors: De facto standard. In: Wikipedia, The Free Encyclopedia. 2020; Retrieved 12:33, October 9, 2020. Reference Source\n\nCost-Benefit analysis for FAIR research data - Policy recommendations. European Commission, Directorate-General for Research and Innovation. 2019. Publisher Full Text\n\nNickerson D, Atalag K, de Bono B, et al.: The Human Physiome: how standards, software and innovative service infrastructures are providing the building blocks to make it achievable. The Royal Society Publishing. Interface Focus. 2016; 6(2): 20150103. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBaker M: How quality control could save your science. Nature. 2016; 529(7587): 456–8. PubMed Abstract \| Publisher Full Text" }	[ { "id": "75752", "date": "01 Feb 2021", "name": "David Nickerson", "expertise": [ "Reviewer Expertise Computational physiology", "reproducibility", "modelling and simulation standards", "model repositories", "semantic annotation." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this opinion article the authors outline many significant scientific advances which are primarily driven by, and possible due to, advances in digital technologies (ICT). They then explore how full realisation of the benefits from these developments depends on open and transparent sharing of knowledge, data, models, etc.\nThe authors approach this discussion from a European Union perspective, in the context of EU policies and projects, but touch of a range of non-EU initiatives. Indeed, the issues addressed are globally relevant and important.\nIn particular, the authors summarise the outcomes from the COST Action CHARME by way of framing the prevalent challenges to adoption of suitable standards in life science research. This is followed by the measures the CHARME network has identified in response to these challenges and to ensure high quality life science research data that facilitates research and innovation.\nThe authors conclude with a vision for a central EU umbrella hub (helpdesk) on standardisation to implement the discussed measures. Such a hub could lead the way with the EU providing global leadership in the adoption of the FAIR principles and standardisation processes and associated services and policies.\nThis reviewer strongly supports the presented measures and the author's goals for improving the reproducibility and FAIRness of the life sciences.\nI only have a few very minor corrections to suggest just to tidy up the article.\nAbstract:\nFAIR Principles.\nIntroduction:\nConsistent use of \"e.g.,\" or \"e.g.\".\n\nPerhaps a missing citation indicated by the \"[citation in the note]\" text?\n\n\"The ignorance of this aspect coms at high for the society, as recently\" - spelling and grammar issues in this sentence.\n\n\"Properly established research standards the alignment of consensus-based best practices, reduce variance, and improve reproducibility and quality in research.\" - this sentence doesn't make sense to me, but not quite sure how to fix it.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [ { "c_id": "6591", "date": "10 May 2021", "name": "Susanne Hollmann", "role": "Author Response", "response": "The authors thank you very much for reviewing and commenting our manuscript. Based on your and the feedback of the other reviewers we prepared a revised version of the manuscript. Consistent use of \"e.g.,\" or \"e.g.” We use now consistently “e.g.” without commats as it refers to the British English style. Perhaps a missing citation indicated by the \"[citation in the note]\" text? The indication for the reference was eliminated. The corresponding reference is given in the consecutive sentence and refers to reference 1: Freedman LP, Inglese J: The increasing urgency for standards in basic biologic research. Cancer Res. 2014; 74(15): 4024–9. \"The ignorance of this aspect coms at high for the society, as recently\" - spelling and grammar issues in this sentence. The sentence has been changed to: “The ignorance of this aspect comes at high costs for the society, as recently stated…” \"Properly established research standards the alignment of consensus-based best practices, reduce variance, and improve reproducibility and quality in research.\" - this sentence doesn't make sense to me, but not quite sure how to fix it. The sentence was changed adding “advance” to complete the sentence: “Properly established research standards advance the alignment of consensus-based best practices, reduce variance, and improve reproducibility and quality in research.”" } ] }, { "id": "77734", "date": "04 Feb 2021", "name": "Laurent Falquet", "expertise": [ "Reviewer Expertise Bioinformatics", "microbial genomics and metagenomics." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors clearly describe the current status of data and tools standards in the Life Sciences. They propose diverse actions to improve and promote the acceptance and usage of standards.\n\nThe manuscript points correctly to the metadata issues for which a big effort must be made to improve the meaningful reuse of the data.\nThere could have been more emphasis on specific issues of medical data where the ethic agreements signed by the patients and the researchers often prevent the open distribution of the data and associated metadata even when anonymized. This a recurring concern affecting the reproducibility of the results.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [ { "c_id": "6592", "date": "10 May 2021", "name": "Susanne Hollmann", "role": "Author Response", "response": "Thank you very much for reviewing and commenting our manuscript and for drawing our attention to this essential aspect. We prepared a revised version of the manuscript based on the feedback of all reviewers. There could have been more emphasis on specific issues of medical data where the ethic agreements signed by the patients and the researchers often prevent the open distribution of the data and associated metadata even when anonymized. This a recurring concern affecting the reproducibility of the results. Under the chapter “Investment and impact” we added the following new paragraph: “The sharing of medical (patient) data is an issue that is highly relevant in the context of Open Science and data sharing but needs to be discussed more intensively elsewhere. Nevertheless, it should be noted here that the strict ethical laws and regulations limit the sharing of patient-derived data – even if anonymised and collated. The availability of these type of resources are a wealth of information that – if made available – could accelerate the research process and avoid duplication of efforts. New paradigms that enforce a participatory approach and active involvement of patients might be a route to solve this problem. Standards are a non-negotiable prerequisite for such an innovative approach.”" } ] }, { "id": "77735", "date": "08 Feb 2021", "name": "Antonella Lanati", "expertise": [ "Reviewer Expertise Quality management in scientific research" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the opinion paper “The need for standardisation in life science research - an approach to excellence and trust”, the authors suggest and discuss measures to ensure high quality and reusability of data in life science research.\nA quality approach, to facilitate research and innovation and to respond to “reproducibility crisis”, is deployed in various aspects and at different levels, from dedicated training and need for Quality Control, up to compliance with international standards and requirements for effective communication. Attention is also paid to the problem of coordination and standardization in research, addressed by quality generation and processing of data, and by adoption of commonly recognized protocols of communications. Particular emphasis is given to the need for recognition and support to free international standards, as the foundation to implement open science principles in research.\nThe paper is very in-depth discussed, wide-ranging and complete, with sharp observations and concrete proposals. Due attention is given not only to the main standardisation needs, but also to specific aspects regarding the stewardship and the management of scientific research.\nThis paper covers the increasing interest for reproducible data and can thus provide a useful contribution to spreading knowledge and principles of research reliability in the scientific community.\nI have no major concerns about the paper, I would suggest only few minor changes:\nAvoid the repetition of few sentences regarding the reproducibility crisis: the topic in page 3, II column, from 14th line on, could be just outlined there and further discussed with the support of citations in page 4, II column, from line 7 on.\n\nThe chapter “education” might benefit from moving after “Reproducibility and quality control” and “Supportive tools and actions”, i. e. after having introduced the problem of the lack of management culture and the need for figures with specific preparation.\n\nThe Measure #3 may include also training on soft skills, as suggested by the needs for education previously highlighted.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [ { "c_id": "6593", "date": "10 May 2021", "name": "Susanne Hollmann", "role": "Author Response", "response": "The authors thank you very much for reviewing and commenting our manuscript. We prepared a revised version of the manuscript based on your and the feedback of the other reviewers. Avoid the repetition of few sentences regarding the reproducibility crisis: the topic in page 3, II column, from 14th line on, could be just outlined there and further discussed with the support of citations in page 4, II column, from line 7 on. Thank you for spotting the repetition in the two instances. We changed the text following your recommendation to: “Reproducibility and quality control The reproducibility crisis as mentioned above2 is another challenge that needs action. Reasons for this reproducibility crisis are manifold, among them the lack of appropriate study design, proper controls or insufficient documentation, etc. But the main reason is the absence of a unifying quality control and assurance framework. High-quality records are essential for the quality and reproducibility of research results and efficient technology transfer. Standards facilitate the alignment of consensus-based best practices, reduce variance, and improve reproducibility in research. The chapter “education” might benefit from moving after “Reproducibility and quality control” and “Supportive tools and actions”, i. e. after having introduced the problem of the lack of management culture and the need for figures with specific preparation. We appreciate the recommendation and moved the chapter “Education and training” and included it after the section on “Supportive tools and actions”. In the chapter “Education and training” we also changed the wording of the first sentence to: “A key challenge is to identify and fill evident gaps in education and training.” The Measure #3 may include also training on soft skills, as suggested by the needs for education previously highlighted. This is an excellent recommendation. We changed the text for measure 3 as follows: “…There are many examples of resistance of researchers to accept rules that QA plans impose and how these resistances can be easily overcome by education11. As indicated above, course programs and educational material on topics like standard operating procedures (SOPs), quality control (QC) and data management need to be developed and implement as part of university curricula and career development modules. Together with “Train-the-Trainer” measures, these educational programs represent a crucial step towards a safe and reliable way to do research.”" } ] } ]	1	https://f1000research.com/articles/9-1398
https://f1000research.com/articles/9-1129/v1	14 Sep 20	{ "type": "Brief Report", "title": "Improving the leptospirosis disease burden assessment by including ambulatory patients from outpatient departments: a cross-sectional study", "authors": [ "Janith Warnasekara", "Parami Aberathna", "Geetha Nanayakkara", "Joseph Vinetz", "Suneth Agampodi", "Parami Aberathna", "Geetha Nanayakkara", "Joseph Vinetz", "Suneth Agampodi" ], "abstract": "Background: In Sri Lanka, the disease burden of leptospirosis is estimated based on a routine notification system, which is predominated by patients ill enough to be hospitalized. The notification system does not function well with ambulatory patients in outpatient departments (OPDs). The objective of this study was to determine the prevalence of leptospirosis in an OPD setting in a regional public hospital in Sri Lanka to provide further estimation of disease burden estimations Methods: This study was conducted in the OPD of the Rathnapura Provincial General Hospital from August to September 2017. Suspected leptospirosis patients were recruited based on standardized criteria and tested using the microscopic agglutination test and quantitative polymerase chain reaction. The number of OPD patients was compared with the reported patient numbers with leptospirosis from the hospital during the same period as the denominator, and the 95% confidence interval was calculated for the proportions using Poisson distribution. Results: During the study period, of 2,960 fever patients presenting to the OPD, 33 (1.1%) were suspected to have leptospirosis; 8/33 suspected (22.3%) cases were confirmed as being due to leptospirosis. There were 82 notifications of leptospirosis cases from hospital inpatients during the same period, none from the OPD. The total missing proportion from the surveillance system was 28.6% (95% CI, 19.4-40.4%). Among OPD patients, 12 (36.4%) had been given antibiotics from a primary care center prior to the OPD visit. No OPD patient was admitted to the hospital for inward care. Conclusions: More than 25% of cases of leptospirosis were not identified because they were not sick enough to be admitted nor subjected to routine leptospirosis diagnostic testing. Antibiotics given without a specific, treatable diagnosis interferes with leptospirosis disease burden assessment. These data have public health implications if the sources of leptospirosis transmission are to be controlled.", "keywords": [ "Leptospirosis", "Sri Lanka", "Outpatient department", "Ambulatory care", "OPD", "burden", "underestimation" ], "content": "Introduction\n\nGenerally speaking, assessing the true burden of disease is required for proper health planning and resource allocation, including the control of transmissible diseases such as leptospirosis. Sri Lankan communicable disease burden estimates are usually done using routinely reported data in the surveillance system1. Lack of actionable diagnostic tests and the diversity of clinical features leading to under-notification of leptospirosis are the major reasons for poor estimation of this disease, a leading cause of acute febrile illness in Sri Lanka2,3. A recently published systematic review has suggested a correction factor for hospitalized leptospirosis cases to more accurately estimate the burden of this disease. This study estimated the incidence of leptospirosis in Sri Lanka as 52.1 per 100,000 population3. However, these estimations and corrections are made for hospitalized patients without considering outpatient departments (OPDs). It is estimated that approximately 5–15% of outpatients with undifferentiated febrile cases could be due to leptospirosis4,5, and undifferentiated febrile patients usually present to OPDs. Finally, these estimates have not been applied to assessing disability-adjusted life years, which is always a challenge for acute febrile illnesses. Therefore, prospective studies in outpatient setting are essential for estimating the burden of disease due to leptospirosis, which in turn is needed to justify investment in diagnostics and vaccine development.\n\nFew studies have assessed leptospirosis in non-hospitalized patients with acute febrile illness. Biggs et al. highlighted the underestimation of leptospirosis due to non-inclusion of ambulatory patients for disease estimates in Tanzania6. A study conducted in Vanuatu showed the importance of screening for leptospirosis among acute febrile illness patients presenting to OPDs during outbreaks, highlighting the need for improved awareness and diagnostic capacity, which are interrelated7. In the Vanuatu study, 12 of 161 (7.4%) suspected patients were confirmed as having leptospirosis. However only 2 of 12 confirmed patients had criteria fulfilling the surveillance case definition, showing the inadequacy of the case definitions used7. Another study conducted in Guadeloupe, Martinique (French territories in the Caribbean) suggested that the actual burden of leptospirosis could be 3 to 4 times higher than reported cases8. A study conducted in Mozambique also provided supportive evidence for the importance of outpatient leptospirosis by estimating that as much as 10% of febrile patients attending ambulatory care could be attributed to leptospirosis9. The purpose of the present study was to determine the prevalence of leptospirosis in an OPD setting in a regional public hospital in Sri Lanka to provide further estimation of disease burden estimations.\n\n\nMethods\n\nThe study was conducted from August 2017 to September 2017 in the OPD of Rathnapura Provincial General Hospital (RPGH) as a part of larger clinico-epidemiological study. Previous data suggested that the Rathnapura district is one of four major districts affected by leptospirosis10. At the time of the present study, the OPD had a separate desk for patients presenting with acute febrile illness. This was partly due to the massive epidemic of dengue ongoing during that period.\n\nOnce the medical officer screened the patients for obvious foci of infection and after sending probable dengue patients for further investigation, a medical graduate awaiting an internship appointment screened the remaining acute undifferentiated fever patients. Clinically suspected patients were recruited as “possible” cases of leptospirosis, using a standardized, written surveillance case definition for Sri Lanka11. In the meantime a survey was conducted among inward clinically confirmed leptospirosis patients of RPGH to assess the past treatment history.\n\nRecruited patients were interviewed using a standardized, written clinical data checklist and a questionnaire (Extended data). A blood sample of 4ml was taken, and 2ml was transferred to a plane tube and 2ml to an EDTA tube and stored in the microbiology laboratory of RPGH.\n\nSamples were transported to the public health research laboratory of the Faculty of Medicine and Allied Sciences, Rajarata University of Sri Lanka. Testing for leptospirosis was done using the microscopic agglutination test (MAT) and quantitative polymerase chain reaction, as previously published in the study protocol12.\n\nHospital notification data were obtained from the infection control unit at RPGH. The number of confirmed OPD patients was compared with the number of leptospirosis-confirmed hospitalized patients during the same period, and normalized to total patient populations. Care-seeking was compared with sample of hospitalized patients treated as leptospirosis by attending physicians.\n\nA SPSS trial version 23 was used for data analysis. A Poisson distribution was used to calculate the 95% confidence interval for the missing patient estimates from OPD.\n\nEthical approval for this research was obtained from the Ethics Review Committee of the Faculty of Medicine and Allied Sciences, Rajarata University of Sri Lanka (No: ERC/2015/18). Written informed consent was obtained from all the patients for participation in the study.\n\n\nResults\n\nA total of 2,960 febrile patients were screened in the fever section of the OPD during the study period. Of these, 33 (1.1%) were clinically suspected leptospirosis patients and all were recruited for the present study (Figure 1). These included 23 (69.7%) men and 10 (30.3%) women. The mean age was 46.5 years (SD 17.1). During the same period, RPGH made 82 notifications of possible cases of leptospirosis from hospitalized patients. The missing OPD patients from the notification accounted for 28.6% (95% CI 19.4-40.4) (Table 1).\n\nOf 33 possible cases, 8 (24.2%) were laboratory confirmed as leptospirosis. One patient was categorized as “probable” with single MAT titre of 1/20012. Of the 33 cases selected, 12 (36.4%) had received treatment from a primary care centre prior to coming to the RPGH OPD. During the same period, we interviewed 29 hospitalized patients who were treated presumptively for leptospirosis. Of these, 19 (66.5%) reported that they were given treatment for fever from a primary care provider prior to hospital admission. However, none of these 19 visited the OPD of RPGH, confirming that the cases presented to OPD are really “missing” from the system.\n\n\nDiscussion\n\nIn this preliminary study to evaluate the missing leptospirosis patient load in the surveillance system, we made three important observations: (1) almost one third of the patients presenting to the OPD of RPGH were missing from the notification system; (2) most of the patients (although we could say none, there might be admissions after the study period) presenting to the OPD were not hospitalized; (3) most of the hospitalized patients sought healthcare from primary care centres rather than from a tertiary care centre. Based on the OPD data, it clearly shows that 28.6% (95% CI 19.4-40.4) of the leptospirosis patients presenting to this tertiary centre were not included in the system. Nevertheless, statistical assumptions cannot be made for the primary care institution without proper studies conducted in local hospitals and private healthcare institutions. This study mainly focused on the cases presenting in an endemic setting and during an outbreak period. The missing numbers can neither be generalized to all areas of Sri Lanka nor for all the months of the year in the same area. Establishing well-functioning disease surveillance system in OPDs and primary care institutions is essential for proper disease burden estimates, not only for leptospirosis, but also for other notifiable diseases. There are various small scale studies that has been conducted to identify feasible methods for disease surveillance, such as incorporating smart phone technology which are being carried by hand by the treating physician13. These feasibility studies need to be up scaled to identify the barriers and the feasible methods to implement the system. Well-planned studies covering outpatient, inpatient and private sector should be initiated for estimating the actual burden of diseases.\n\n\nData availability\n\nZenodo: OPD Lepto Data base - Clinical check List, http://doi.org/10.5281/zenodo.401324814.\n\nZenodo: OPD Lepto Data base - Clinical check List, http://doi.org/10.5281/zenodo.401324814.\n\nThis project contains the following extended data:\n\n- Questionnaire OPD (1st Interview)\n\n- Event calendar\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nEpidemiology Unit - Sri Lanka: Quarterly Epidemiological Report. Accessed June 27, 2017. Reference Source\n\nDahanayaka NJ, Warnasekara YP, Rajapakse RM, et al.: Validity of Lateral Flow Immunochromatographic-Assays (LFIA) in diagnosis of leptospirosis. Ceylon Med J. 2017; 62(4): 248–49. PubMed Abstract \| Publisher Full Text\n\nWarnasekara J, Koralegedara I, Agampodi S: Estimating the burden of leptospirosis in Sri Lanka; a systematic review. BMC Infect Dis. 2019; 19(1): 119. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbhilash KPP, Jeevan JA, Mitra S, et al.: Acute undifferentiated febrile illness in patients presenting to a Tertiary Care Hospital in South India: clinical spectrum and outcome. J Glob Infect Dis. 2016; 8(4): 147–154. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nReller ME, Bodinayake C, Nagahawatte A, et al.: Leptospirosis as frequent cause of acute febrile illness in southern Sri Lanka. Emerg Infect Dis. 2011; 17(9): 1678–1684. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBiggs HM, Hertz JT, Munishi OM, et al.: Estimating leptospirosis incidence using hospital-based surveillance and a population-based health care utilization survey in Tanzania. PLoS Negl Trop Dis. 2013; 7(12): e2589. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPakoa JG, Soupé-Gilbert ME, Girault D, et al.: High incidence of leptospirosis in an observational study of hospital outpatients in Vanuatu highlights the need for improved awareness and diagnostic capacities. PLoS Negl Trop Dis. 2018; 12(6): e0006564. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCassadou S, Rosine J, Flamand C, et al.: Underestimation of Leptospirosis Incidence in the French West Indies. Foley J ed. PLoS Negl Trop Dis. 2016; 10(4): e0004668. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCollares-Pereira M, Gomes AC, Prassad M, et al.: Preliminary survey of Leptospirosis and Lyme disease amongst febrile patients attending community hospital ambulatory care in Maputo, Mozambique. Cent Afr J Med. 1997; 43(8): 234–238. Accessed October 13, 2019. PubMed Abstract\n\nWarnasekara JN, Agampodi S: Leptospirosis in Sri Lanka. Sri Lankan J Infect Dis. 2017; 7(2): 67.Publisher Full Text\n\nAgampodi S: Case definitions in Leptospirosis: a note to Sri Lankan researchers. Sri Lankan J Infect Dis. 2012; 2(2): 55–57. Publisher Full Text\n\nAgampodi S, Warnasekara J, Jayasundara D, et al.: Study protocol: characterising the clinical, epidemiological and aetiological aspects of leptospirosis in Sri Lanka: a hospital based clinico-epidemiological study. BMJ Open. 2019; 9(9): e027850. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWarnasekara YPJN, Agampodi SB, Marage PM, et al.: Real time surveillance of dengue fever – a preliminary study. Ceylon Med J. 2016; 61(4): 197–198. PubMed Abstract \| Publisher Full Text\n\nWarnasekara J, Agampodi S: OPD Lepto Data base - Clinical check List. Zenodo. 2020. http://www.doi.org/10.5281/zenodo.4013248" }	[ { "id": "74988", "date": "10 Feb 2021", "name": "Georgies F Mgode", "expertise": [ "Reviewer Expertise Rodent borne zoonotic diseases - leptospirosis and plague disease", "tuberculosis and taxonomy" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present important findings on leptospirosis from outpatients population. It is a well designed study and covered a large sample size consisting of 2,960 individuals with febrile illness.\n\nIn the methodology section: plane tube should be \"plain tube\".\n\nFurther description of the microscopic agglutination test (MAT) is needed given that it is a core test for confirmation of leptospirosis. Information on Leptospira serovars used, age and whether was live or killed is much needed.\n\nRecommendation to establish a well-functioning disease surveillance system is important.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6348", "date": "11 Feb 2021", "name": "Janith Warnasekara", "role": "Author Response", "response": "Thank you very much for the response. I agree with the comments made by the reviewer. I would like to do the following changes in the manuscript. Change the plane into plain in methodology Add a supplementary file of full MAT protocol Thank you very much" } ] }, { "id": "82774", "date": "19 Apr 2021", "name": "Chinthika P. Gunasekara", "expertise": [ "Reviewer Expertise Leptospirosis", "immune response" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this study the authors address an important aspect of leptospirosis burden in Sri Lanka that is a timely need. The main finding of the study that is conducted in a high prevalent district Rathnapura is that the true burden of leptospirosis is underestimated and about 28% cases coming to the OPD for treatment are not reported. I suggest that the authors include more recent references including data from the epidemiological unit as there are several publications done in Sri Lanka published more recently. The supporting files are missing some components as the part C of the questionaire. Further the abstract results section mentions some data that is not described by way of table, additional data or described in the results section and this should be corrected to support the findings and conclusion. for example antibiotic history. There are several spelling errors Eg : plane tube - plain tube. Overall I believe that the results section should be slightly expanded to support the conclusions and findings. This is an interesting paper and I recommend it for indexing subject to revision.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6613", "date": "11 May 2021", "name": "Janith Warnasekara", "role": "Author Response", "response": "Comment I suggest that the authors include more recent references including data from the epidemiological unit as there are several publications done in Sri Lanka published more recently. Reply This was submitted as a brief report with a specific focus and the reference included were only directly related to the narrow focus. This was the reason for having a small results section. Extensive discussions on all Sri Lankan publications were done as a part of the larger study, which has included epidemiological unit data and data from all recent papers. (Published systematic review DOI: 10.1186/s12879-018-3655-y 6364467 Comment The supporting files are missing some components as the part C of the questionaire. Reply Yes. We understand that some of the phrases in questionnaire are mis leading. This study was conducted as a part of large clinical study related to leptospirosis. The Part C and further sections were for inward patients (not relevant to this brief report). We only applied Part A and B for OPD patients as the duration of interview with an OPD patient is less than the inward patients. To avoid misinterpretation, misleading parts were explained in the questionnaire. Corrected questionnaire is uploaded as extended data. Eg: Go to part C (Only the inward Patients) Comment Further the abstract results section mentions some data that is not described by way of table, additional data or described in the results section and this should be corrected to support the findings and conclusion. for example antibiotic history. Reply First part of the results section of the abstract was explained in the figure one and the rest is in the table. We identify that 12 patients received antibiotics are mentioned as “antibiotics” in the abstract and as “treatment” in the main text. To avoid misinterpretation, we have changed the treatment as “antibiotic treatment” in the main text results section. Comment There are several spelling errors Eg: plane tube - plain tube. Reply Spelling, grammar and formatting of the text are revised and corrected. All changes are mentioned as track changes in the manuscript. Comment Overall I believe that the results section should be slightly expanded to support the conclusions and findings. Reply We humbly agree that the results section is small. As explained earlier, this is a brief report with very specific focus and we have included all the data relevant to this aim it this paper." } ] } ]	1	https://f1000research.com/articles/9-1129
https://f1000research.com/articles/9-616/v1	16 Jun 20	{ "type": "Research Article", "title": "Mid-level health providers for primary healthcare: a rapid evidence synthesis", "authors": [ "Sandeep Moola", "Soumyadeep Bhaumik", "Devaki Nambiar", "Soumyadeep Bhaumik", "Devaki Nambiar" ], "abstract": "Background: Health care services, in many countries, are increasingly being provided by cadres not trained as physicians, but capable of performing several diagnostic and clinical functions. These substitute health workers are referred to as mid-level health providers (MLHPs). The health and wellness centres under India’s Comprehensive Primary Health Care programme have teams led by MLHPs who can aid doctors. The objective of this study was to rapidly synthesise evidence on the effectiveness of MLHPs for primary health care. Methods: The review team undertook a rapid overview of systematic reviews that compared MLHPs with doctors and different types of MLHPs involved in the delivery of health care were included, with a perspective on low- and middle-income countries, including India. Results: Seven systematic reviews were included in the final report. Mortality outcomes in relation to pregnancy and childbirth care services showed no significant differences in care provided by MLHPs when compared with doctors. Pregnancy care provided by midwives was found to slightly improve quality of care when compared to care delivered by doctors. The risk of failure or incomplete abortion for surgical abortion procedures provided by MLHPs was twice when compared to the procedures provided by doctors. Moderate to high certainty evidence showed that initiation and maintenance of antiretroviral therapy for HIV-infected patients by a nurse or clinical officer slightly reduced mortality. High certainty evidence showed that chronic disease management by non-medical prescribers reduced some important physiological measures when compared to medical prescribing by doctors. Conclusions: To date, this is the first rapid overview of evidence on MLHPs. Evidence suggests that MLHPs might be suitable to deliver quality care in certain areas of health and they may be relevant and feasible in countries like India. However, the roles and subsequent training and regulation of MLHPs might be different for different care domains.", "keywords": [ "Rapid review", "rapid evidence synthesis", "systematic reviews", "mid-level health providers", "MLHPs", "low- and middle-income countries" ], "content": "Introduction\n\nThere is a growing momentum worldwide to improve access to healthcare and provide efficient and cost-effective primary health care (PHC)1. Mid-level health providers (MLHPs) are currently being used in high- and low-income countries to assist doctors and specialists and/or to render services independently, particularly in resource-poor settings to make up for the scarcity of health professionals. A cornerstone of current health systems reform efforts in India is the flagship Ayushman Bharat (AB) program. Largely the program has an insurance component (Pradhan Mantri Jan Arogya Yojana, PMJAY) and development of Health and Wellness Centres (HWCs) as strategies to advance on the path to universal health coverage2.\n\nAyushman Bharat’s HWC sub-strategy, the comprehensive primary health care (CPHC), conceives of MLHPs as a key focal point for service organisation and delivery, performing a range of screening, diagnostic and clinical functions and improve health systems at the frontline. The program conceptualises 12 different packages for the CPHC reforms3. One key pillar of rolling out the AB-HWC component is the inclusion of new health cadre trained and accredited for a set of skills/competencies related to PHC and public health. Further, one of the aims of this programme is the transformation of existing sub-health centres and PHCs to HWCs, with teams led by MLHPs.\n\nThe National Health Systems Resource Centre (NHSRC), the technical support agency of the National Health Mission, is responsible for developing the curriculum for MLHPs. We received a request from the NHSRC for a rapid review of evidence on the effectiveness of MLHPs in the PHC context of low- and middle-income countries (LMICs) to understand the role MLHPs can play in different packages. We host a rapid evidence synthesis (RES) platform, which provides gratis RES to public agencies. RES or rapid review is an emerging form of evidence synthesis that is increasingly being promoted by the WHO and employed by governments to inform decision making4. We thus synthesised evidence related to the effectiveness of MLHPs in the PHC context of LMICs.\n\n\nMethods\n\nWe conducted a rapid overview of systematic reviews (SRs) of evidence on the effectiveness of MLHPs within a span of about eight weeks and in all domains corresponding to the CPHC package in Ayushman Bharat. The 12 CPHC packages are: pregnancy and childbirth; neonatal and infant health services; childhood and adolescent health services; family planning, contraceptive services and other reproductive care services; communicable diseases (prevention and management); non-communicable diseases; elderly and palliative care; oral health care; ophthalmic and ear, nose and throat (ENT) care; mental health and emergency medical services3.\n\nThe World Health Organization (WHO), defined MLHP as “a health provider who is trained, authorised and regulated to work autonomously, receives pre-service training at a higher education institution for at least 2–3 years and whose scope of practice includes (but is not restricted to) being able to diagnose, manage and treat illness, disease, and impairments (including performing surgery, where appropriately trained), prescribe medicines, as well as engage in preventive and promotive care”1, p.8. However, MLHPs in various countries have been variously referred to as substitute health workers, auxiliaries, non-physician clinicians, and include cadres such as clinical officers, medical assistants, physician assistants, nurse practitioners, and surgical technicians. Institutions and researchers worldwide use alternate or less well-specified definitions, and therefore MLHP as defined in the SRs was considered for this review. Therefore, we used broad criteria for the rapid overview wherein we accepted the definition of MLHPs as defined by the SR authors. The overview of SRs is an appropriate study design for our research because we intended to summarise the evidence for multiple conditions in different disease/condition domains for the same type of intervention and on similar health systems, clinical and public health outcomes.\n\nParticipants. The RES considered SRs assessing outcomes for participants receiving care from MLHPs in LMICs, including India.\n\nIntervention and comparators. SRs that compared service delivery provided by MLHPs with doctors or other types of MLHPs were included. The MLHPs included were midwives, nurses, auxiliary nurses, nurse assistants, non‐physician clinicians, and surgical technicians.\n\nOutcomes. The following outcomes were considered for inclusion based on the initial discussions with the requester: healthcare and clinical outcomes (mortality, morbidity, outcomes associated with care delivery, and physiological measures); access to care; and quality of care (including patient or client satisfaction with care).\n\nStudy design. SRs including studies of any quantitative study design, irrespective of whether they have or have not conducted meta-analyses and irrespective of whether they have or have not used the Grading of Recommendations Assessment, Development and Evaluation (GRADE) framework to assess the certainty of evidence were included. Qualitative SRs were not considered.\n\nContext. SRs with a focus on and/or including studies from LMICs and India were included.\n\nGiven time constraints, the search was limited to published and indexed articles, and those published in the English language. The following databases were searched (from database inception up until March 2019): Cochrane Database of Systematic Reviews; Medline (PubMed); EMBASE; Health Systems Evidence; and CINAHL. An additional search was conducted from April 2019 to April 2020 to update the review findings for recency and relevancy. Search strategies (for both the periods) are provided separately for each database (see Extended data)5.\n\nThe lead reviewer (SM) independently screened the titles and abstracts of studies for inclusion, following which full-text examination of eligible studies was conducted for potential inclusion. A second reviewer (SB) randomly verified the results of the study selection process during both the screening stages. For each domain of interest, where multiple SRs were available, only one SR was included based on its comprehensiveness, recency, and quality. Each SR was independently assessed for methodological quality by using established standardised criteria (A MeaSurement Tool to Assess systematic Reviews (AMSTAR) 2 checklist)6. Data from included reviews was extracted using a pre-defined template, which included variables such as review type, review question, countries/settings, participants characteristics, interventions, outcome measures and review conclusions. The lead author (SM) independently extracted all relevant outcome data, with random verification of 20% of the included studies by another author (SB).\n\nThe GRADE approach was used to assess the certainty of the evidence using a transparent framework for developing and presenting the summary of findings tables7–10. The GRADE of evidence was synthesised with respect to a PHC setting and in an LMIC context to make the product locally relevant9,10.\n\nAs part of the RES process, the RES team and NHSRC jointly convened a policy dialogue to engage and consult with relevant stakeholders to present an interim draft of the MLHP policy brief. The stakeholders included policy makers (key stakeholders from government agencies and collaborators), health system managers, and researchers from more than eight states in India.\n\n\nResults\n\nThe search for evidence identified 5171 studies (Figure 1 – Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) flow diagram). Following the study screening process, full-text articles were retrieved for 30 potentially relevant studies. In cases where there were multiple SRs for the same domain, the SR that was the most recent and provided comprehensive information (as per authors’ (SM, SB) consensus) was selected and included. Following full-text examination, 23 out of 30 SRs were excluded. An additional 717 records were identified in an updated search. However, following the study selection process, none of the reviews were found to be relevant to the topic of interest (Figure 2 – PRISMA flow diagram (updated search)). Overall, seven SRs were included in the RES.\n\nSearch conducted from database/s inception up until March 2019.\n\nUpdated search from April 2019 to April 2020.\n\nThe majority of the studies included in the SRs were randomised controlled trials (RCTs), with some quasi-experimental study designs and observational studies. Key characteristics of the included SRs are provided in the Extended data file5. All SRs, except one11 included studies that were mostly conducted in LMICs. The studies related to HIV/AIDS were mostly conducted in sub-Saharan African countries. Most studies compared care provided by midwives or auxiliary nurse midwives or nurses with that provided by doctors working in a team along with midwives or nurses.\n\nThe AMSTAR-2 checklist6 was used to assess the methodological quality of SRs included in the report. The checklist is a 16-item questionnaire. The critical appraisal results of the included SRs are provided in the Extended data file5. Six out of seven SRs were of moderate to high methodological quality and well reported. Almost all the SRs did not refer to a priori protocol and publication bias was not assessed. One SR by Chaudhary et al. was of poor quality, as assessed by the checklist12.\n\nKey findings. The key findings from the included SRs have been categorised based on the various healthcare domains of interest in the CPHC package3. The quality of evidence for the main outcomes is summarised using the GRADE approach and ‘Summary of Findings’ tables7–10. The Summary of Findings tables aid in recording results, outcomes, and outcome risks in a structured synthesis format.\n\nMLHPs for care in pregnancy and childbirth. An SR compared the effectiveness of care provided by MLHPs, particularly midwives and auxiliary nurse midwives with doctors providing care in a team with midwives11. The review included patients receiving pregnancy and childbirth services including antenatal care. The majority of the studies were conducted in tertiary care settings and developed countries. Most of the evidence was assessed as low certainty. It was found that the use of intrapartum analgesia and episiotomies were less likely with care provided by midwives when compared with that provided by doctors working along with midwives. Also, no significant difference in rates for performing caesarean section, postpartum haemorrhage, and preterm births were reported.\n\nNo significant difference in the likelihood of an incomplete abortion was reported between groups of patients treated by auxiliary nurse midwives compared to those cared for by doctors. However, the likelihood of a complication during or an adverse event after manual vacuum aspiration was significantly greater with care provided by auxiliary nurse midwives. There was very low certainty evidence to suggest that pregnancy care provided by clinical officers reduced the likelihood of early neonatal death or postoperative maternal health outcomes, such as fever and wound infections. Table 1 provides a summary of findings and certainty of evidence related to pregnancy and childbirth care provided by midwives, auxiliary nurse midwives and clinical officers with that provided by doctors in a team with midwives.\n\n1Downgraded one level due to serious risk of bias and another two levels due to indirectness (almost all the studies were conducted in tertiary care centres and high-income countries).\n\n2Downgraded one level due to serious inconsistency (considerable heterogeneity was found).\n\n3Downgraded one level due to imprecision (single study with a small sample size yielding wide confidence intervals spanning line of no effect).\n\n4Quality of evidence was downgraded from Low (observational study design) to Very low due to a very serious risk of bias.\n\nCI, confidence interval; GRADE, Grading of Recommendations, Assessment, Development and Evaluations; RR, risk ratio; RCT, randomised controlled trial; QoC, quality of care; MLHPs, mid-level health providers.\n\nMLHPs for neonatal and infant health care services. The effectiveness of midwives/nurses delivering care for neonatal and infant healthcare services was compared with that provided by doctors or obstetricians in a team with midwives in a SR11. The population included patients receiving neonatal and infant health services. The majority of the studies were conducted in tertiary care settings and developed countries. The certainty of the evidence was assessed as low quality. The review results showed that there was no significant difference between the groups in foetal or neonatal death rates. None of the studies included in the review reported on clinical outcomes, and outcomes related to quality of care and access to care. Table 2 presents the review findings in plain language format and the certainty of the evidence for the relevant outcome.\n\n1Downgraded one level due to serious risk of bias and two levels due to indirectness (almost all the studies were conducted in tertiary care centres).\n\nCI, confidence interval; GRADE, Grading of Recommendations, Assessment, Development and Evaluations; RR, risk ratio; RCT, randomised controlled trial; MLHPs, mid-level health providers.\n\nMLHPs for family planning, contraceptive and other reproductive health care services. Another SR by Barnard et al. evaluated the safety and effectiveness of surgical and medical abortion procedures administered by MLHPs compared to doctors13. The review included various MLHPs who included nurses, midwives, doctor assistants, and physician assistants delivering care for patients requesting abortion procedures, either surgical or medical. The majority of the studies were conducted in PHC settings and LMICs. Much of the evidence was of low or very low quality. The review found that the evidence for surgical abortion procedures provided by MLHPs was lacking. Further, evidence from cohort studies suggested that there was an increase in the risk of failure or incomplete abortion for surgical abortion procedures when provided by MLHPs. However, no statistically significant differences in complications alone, immediate complications or delayed complications were reported when surgical abortion was provided by MLHPs. Concerning medical abortion procedures, the review results suggested MLHPs could safely and effectively carry out these procedures. No significant differences were reported for abortion failure or incomplete abortion. None of the studies included in the SR examined other outcomes of interest such as mortality, quality of care, and access to care. Table 3 presents a summary of findings on various outcomes related to surgical and medical abortion procedures provided by MLHPs compared to doctors.\n\n*Total complications - incomplete or failed abortion and complications\n\n1Downgraded one level due to imprecision and additional one level due to indirectness as studies included were not from the primary healthcare context.\n\n2Downgraded two levels due to risk of bias and one level for imprecision (wide confidence intervals)\n\n3Downgraded one level due to serious risk of bias\n\nCI, confidence interval; GRADE, Grading of Recommendations, Assessment, Development and Evaluations; RR, risk ratio; RCT, randomised controlled trial; MLHPs, mid-level health providers.\n\nMLHPs for communicable diseases. Two SRs examined the effectiveness of the delivery of antiretroviral therapy (ART) provided by MLHPs in HIV-infected patients14,12. The reviews included studies mainly conducted in primary health care settings and LMICs. The studies included in the reviews compared ART provided by nurses or clinical officers with doctors. The certainty of the evidence varied for different outcomes, from high to very low quality. However, the evidence for various outcomes was based on relatively few studies. The review reported that there was no significant difference in mortality, with lower rates of losses to follow up at 12 months. Further, no difference in death or number of patients lost to follow up at 12 months was reported when doctors initiated therapy and nurses provided follow-up. The reviews suggested that shifting tasks from doctors to MLHPs may help in potentially reducing costs of ART provision, without compromising on the quality of care and patient outcomes. Table 4 provides a summary of findings reported in the SRs for outcomes related to the initiation and maintenance of ART in HIV-infected patients.\n\n1 Downgraded by one level for imprecision due to a wide confidence interval\n\n2 Rated low because of observational study designs. Not downgraded for risk of bias\n\n3 Downgraded by one level for imprecision due to low event numbers\n\nCI, confidence interval; GRADE, Grading of Recommendations, Assessment, Development and Evaluations; RR, risk ratio; RCT, randomised controlled trial; MLHPs, mid-level health providers; HIV, human immunodeficiency virus; AIDS, acquired immunodeficiency syndrome; ART, antiretroviral therapy.\n\nMLHPs for non-communicable diseases. Two reviews compared the effectiveness of care provided by non-physician health workers (NPHWs) for patients with non-communicable diseases in primary and secondary health care settings15,16. The NPHWs included nurses, pharmacists, allied health professionals, and physician assistants. The care provided by NPHWs was compared to that provided by doctors for various physiological measure outcomes, health-related quality of life, and access to care. The evidence assessed was of moderate to high quality. The findings from the two reviews suggested that care provided by NPHWs with varying but high degrees of autonomy and with support was comparable to that provided by doctors for various relevant outcomes. Care prescription by NPHWs significantly improved outcomes such as systolic blood pressure, glycated haemoglobin and low-density lipoprotein levels. Also, the care provided by NPHWs improved health-related quality of life (physical component). However, the mental health-related quality of life was reduced with the care provided by NPHWs compared to that provided by doctors. There was a lack of conclusive evidence on outcomes related to access to care. Table 5 presents a summary of findings for various relevant outcomes related to chronic diseases.\n\n1Downgraded one level due to serious inconsistency (considerable heterogeneity was found)\n\n2Downgraded one level due to indirectness (prescribing component effect on quality of life difficult to determine)\n\nCI, confidence interval; GRADE, Grading of Recommendations, Assessment, Development and Evaluations; MD, mean difference; RCT, randomised controlled trial; NPHW, non-physician health worker.\n\nMLHPs for mental health. One SR compared the effectiveness of delivery of care provided by non-specialist health workers (NSHWs) to that provided by mental health specialists in women with perinatal depression17. The NSHWs included midwives, nurses, and community health workers. The studies included in the review were conducted in primary health settings and LMICs. The review found that the NSHWs could effectively deliver psychological interventions for perinatal depression in low-resource settings, particularly where specialist services are both scarce and expensive. The review did not examine other relevant outcomes such as mortality, quality of care, and access to care. The review lacked proper reporting and hence it was not possible to assess the certainty of evidence by GRADE. The SR included nine RCTs involving a total of 14,555 participants. Table 6 briefly presents a narrative summary of the findings reported in the review.\n\nNSHW, non-specialist health worker; EPDS, Edinburgh Postnatal Depression Scale; CES-D, Center for Epidemiological Studies Depression Scale; BDI, Beck Depression Inventory; GHQ, General Health Questionnaire; HDRS, Hamilton Depression Rating Scale; GRADE, Grading of Recommendations, Assessment, Development and Evaluations; SR, systematic review.\n\nMLHPs for other packages of care. This RES did not identify any SRs that assessed the role of MLHPs in the provision of following health services.\n\nMLHPs for childhood and adolescent health services\n\nMLHPs for ophthalmic and ENT conditions\n\nMLHPs for elderly and palliative health care\n\nMLHPs for emergency medical services\n\n\nDiscussion\n\nIn this rapid overview of SRs, we examined the evidence on the effectiveness of care provided by MLHPs in LMICs for various healthcare domains of India’s CPHC package3, and contextualised the certainty using GRADE approach7. We found that there is some evidence that MLHP-led (auxiliary nurses and midwives, physician assistants, non-physician health workers, and community health workers) care may be appropriate in patients for management of various outcomes in different healthcare domains of interest such as maternal and child health, neonatal and infant health, and communicable and non-communicable disease management when compared to physician or doctor-led care, but the certainty of the evidence for this was mostly low or moderate (barring a few exceptions). As such, while MLHPs can be considered as an alternative to medical professionals for some domains, the certainty of evidence implies the need for building an evidence base and careful evaluation of programs as they are scaled up in India.\n\nThere is, however, no synthesised evidence in the form of SRs for childhood and adolescent health services, ophthalmic and ENT conditions, elderly and palliative health care, or emergency medical services. There is a need for conducting well-designed primary studies on these domains to inform future plans for rolling out of MLHPs for CPHC implementation in India.\n\nWhile we looked at global evidence, the use of GRADE enabled us to contextualise evidence to India. A robust, transparent and comprehensive search strategy was utilised to identify all relevant SRs; and methodological quality assessment of included SRs was conducted using a standardised checklist. Having a wide scope covering multiple CPHC domains enabled the identification of knowledge gaps that could inform relevant stakeholders at the national and state levels.\n\nAs part of the RES process, we presented the interim policy brief to engage with key stakeholders, seeking to ensure that the product was robust, relevant, and useful to the target audience. The stakeholders deliberated on the policy brief and provided feedback on the usefulness, relevance, format, and the use of GRADE. Following deliberations with the stakeholders, several changes were made to the policy brief in terms of the use of standardised definitions, the use of more plain language statements, and contextualising evidence to the Indian setting. The inclusion of SRs provided more high-level insight into synthesised evidence around MLHPs. We did not update the reviews and as such we acknowledge the limitation of evidence from primary studies that may have not been included in the reviews.\n\nThe use of the WHO definition for MLHP was in alignment with the MLHP term described in the Ayushman Bharat operational guidelines on CPHC. The search for and comparison of global evidence fit the inclusion criteria that were developed in consultation with the requester. The definition of MLHPs as defined by the WHO and in most countries (which we synthesised evidence from), does not include cadres like Ayurvedic, Homoeopathic, Siddha, and other complementary and alternative medicine practitioners who are part of the MLHP programme in India. As such, this limits generalisation and underlines the need for high-quality studies to be conducted in India to assess the effect these workers have, relative to others, in achieving desired outcomes.\n\nWe found several gaps in current research on MLHPs. Evidence from SRs of randomised controlled trials is important, but this approach may not be the most appropriate, as they are unlikely to yield data to inform such a complex intervention. Further, substantial primary research may be required on outcomes related to access to care and quality of care. Future studies may consider addressing the implementation aspects as part of the existing healthcare system as well as the cost-effectiveness in LMICs.\n\nThere is limited evidence on strategies and facilitators for the implementation of universal healthcare policies and the provision of equitable health care through MLHPs in India. A study in Chhattisgarh that assessed the clinical competence of non-physician clinicians and physicians in the delivery of primary health-care services found comparable levels of competency18. Another study conducted in Chhattisgarh reported that physicians and nonphysician clinicians performed similarly in terms of patient satisfaction, trust, and perceived quality19. In Assam, a three-year rural health practitioner course was developed and implemented to select, train and deploy Rural Health Practitioners (RMPs, a type of MLHP) in sub-centres, which showed significant improvements in the number and the range of services delivered20.\n\n\nConclusion\n\nIn conclusion and based on our findings, utilisation of MLHPs for care provision for certain healthcare domains may be applicable, relevant, and feasible in LMICs, including in India. MLHPs such as nurse practitioners, physician assistants, and community health officers will be required for primary care to fill the gaps in access and quality in health services. However, the roles and subsequent training and regulation of MLHPs might be different for several CPHC packages and there is a need for embedded research and robust evaluations in the future.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nFigshare: Extended data.docx. https://doi.org/10.6084/m9.figshare.12401525.v15\n\nThis project contains the following extended data:\n\n- Appendix 1: Search strategies (since database inception up until March 2019)\n\n- Appendix 2: Updated search strategies (April 2019 to April 2020)\n\n- Appendix 3: Key characteristics of the included SRs\n\n- Appendix 4: Critical appraisal results of included systematic reviews assessed using the AMSTAR-2 checklist\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nWorld Health Organization (WHO): Mid-level health providers: a promising resource to achieve the health Millennium Development Goals. 2010. 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PubMed Abstract \| Publisher Full Text\n\nLassi ZS, Cometto G, Huicho L, et al.: Quality of care provided by mid-level health workers: systematic review and meta-analysis. Bull World Health Organ. 2013; 91(11): 824–833I. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMdege ND, Chindove S, Ali S: The effectiveness and cost implications of task-shifting in the delivery of antiretroviral therapy to HIV-infected patients: a systematic review. Health Policy Plan. 2013; 28(3): 223–36. PubMed Abstract \| Publisher Full Text\n\nBarnard S, Kim C, Park MH, et al.: Doctors or mid-level providers for abortion. Cochrane Database Syst Rev. 2015; (7): CD011242. PubMed Abstract \| Publisher Full Text\n\nKredo T, Adeniyi FB, Bateganya M, et al.: Task shifting from doctors to non‐doctors for initiation and maintenance of antiretroviral therapy. Cochrane Database Syst Rev. 2014; (7): CD007331. PubMed Abstract \| Publisher Full Text\n\nJoshi R, Alim M, Kengne AP, et al.: Task shifting for non-communicable disease management in low and middle income countries--a systematic review. PLoS One. 2014; 9(8): e103754. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWeeks G, George J, Maclure K, et al.: Non-medical prescribing versus medical prescribing for acute and chronic disease management in primary and secondary care. Cochrane Database Syst Rev. 2016; 11(11): CD011227. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChowdhary N, Sikander S, Atif N, et al.: The content and delivery of psychological interventions for perinatal depression by non-specialist health workers in low and middle income countries: a systematic review. Best Practice Res Clin Obstet Gynaecol. 2014; 28(1): 113–33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRao KD, Sundararaman T, Bhatnagar A, et al.: Which doctor for primary health care? Quality of care and non-physician clinicians in India. Soc Sci Med. 2013; 84: 30–4. PubMed Abstract \| Publisher Full Text\n\nRao KD, Stierman E, Bhatnagar A, et al.: As good as physicians: patient perceptions of physicians and non-physician clinicians in rural primary health centers in India. Glob Health Sci Pract. 2013; 1(3): 397–406. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNational Health Systems Resource Centre: Rural Health Practitioners in Assam: Mid-Level Care Provider for Comprehensive Service Delivery in Sub Centers. NHSRC; 2014. Reference Source" }	[ { "id": "76144", "date": "18 Jan 2021", "name": "Shradha S. Parsekar", "expertise": [ "Reviewer Expertise Epidemiology", "Overview of systematic reviews", "Qualitative research", "Public Health", "Systematic review" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for providing me opportunity to review this piece of work. Considering the lack of qualified doctors in some of the resource limited settings like India, mid-levels health providers can be an alternative. I must appreciate authors for this work and presenting the findings in a comprehensive manner.\n\nI have minor comments:\n\nThe focus of the rapid overview of systematic review was on LMICs, however some of the SRs included studies conducted in high income countries (e.g., Barnard et al., 20151 included 50% studies from HICs, Weeks et al., 20162 included 42 of 46 studies conducted in HICs, similarly, Lassi et al., 20133 included studies majorly conducted in HICs). Hence, it would be good if the authors make it clear in the inclusion criteria > context- what percent of included studies within SRs should have been conducted in LMICs. Or explicitly state, the evidence from HICs were eligible considering the statement reported in Discussion section, “While we looked at global evidence….”. Secondly, in the result section it was reported, “All SRs, except one11 included studies that were mostly conducted in LMICs”. However, SR by Weeks et al., 2016 also included studies majorly conducted in HICs.\n\nIn the Discussion section it was reported, \"While we looked at global evidence, the use of GRADE enabled us to contextualise evidence to India.\" Although GRADE help in certainty of evidence, it is worthwhile to consider the contextual factors while contextualising the evidence to India, which I understand was not the focus of this overview of systematic review.\n\nReference numbering in the extended file and the main text do not match, kindly make necessary edits. E.g., Barnard et al., 2015 is reference number 13 in main text while in supplementary file it is 11.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6417", "date": "10 May 2021", "name": "Sandeep Moola", "role": "Author Response", "response": "Dear Reviewer, Thank you for taking out time to review the paper and for your valuable comments. Sincere apologies for the delay in replying. Will revise and upload a new version of the article based on your feedback." }, { "c_id": "6617", "date": "10 May 2021", "name": "Sandeep Moola", "role": "Author Response", "response": "The focus of the rapid overview of systematic review was on LMICs, however some of the SRs included studies conducted in high income countries (e.g., Barnard et al., 20151 included 50% studies from HICs, Weeks et al., 20162 included 42 of 46 studies conducted in HICs, similarly, Lassi et al., 20133 included studies majorly conducted in HICs). Hence, it would be good if the authors make it clear in the inclusion criteria > context- what percent of included studies within SRs should have been conducted in LMICs. Or explicitly state, the evidence from HICs were eligible considering the statement reported in Discussion section, “While we looked at global evidence….”. Thank you for raising this point. We have included a statement, which was initially missing, to accurately reflect the nature of the included studies in the review. Secondly, in the result section it was reported, “All SRs, except one11 included studies that were mostly conducted in LMICs”. However, SR by Weeks et al., 2016 also included studies majorly conducted in HICs. We have deleted the statement. In the Discussion section it was reported, \"While we looked at global evidence, the use of GRADE enabled us to contextualise evidence to India.\" Although GRADE help in certainty of evidence, it is worthwhile to consider the contextual factors while contextualising the evidence to India, which I understand was not the focus of this overview of systematic review. We added relevant information to provide more clarity (page 22) Reference numbering in the extended file and the main text do not match, kindly make necessary edits. E.g., Barnard et al., 2015 is reference number 13 in main text while in supplementary file it is 11. Thank you for pointing out the error. We checked the main text and the extended data file and fixed the referencing numbering errors that were seen in a couple of places." } ] }, { "id": "81670", "date": "31 Mar 2021", "name": "Guido Bendezu-Quispe", "expertise": [ "Reviewer Expertise Public health", "epidemiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAbstract (Introduction: Provide only necessary information to inform the context of the study. Methods: There is insufficient information about the study methods (quality of the included studies, stakeholder engagement, others). Results: no results are presented on the evaluation of the quality of the evidence. Conclusion: The conclusion does not emphasize that the evidence is of low or moderate quality on the usefulness of MLHPs in primary healthcare, and this should be mentioned).\n\nIntroduction: The need for a rapid evidence synthesis for the study topic is not explicitly justified. I believe that the word \"gratis\" in the last paragraph is not suitable. The introduction is focused on India. This topic is suitable for LMIC. Hence, I recommend focusing the narrative of the manuscript on LMIC.\n\nMethods: Specify stakeholder participation. The methodology describes that a single reviewer performed the screening process (specify the reason for this).\nResults: Specify characteristics of the documents included in the analysis. There are tables that could be merged (table 2 to 6) to reduce the number of appendices since the last ones include data that should be presented in the manuscript body.\nDiscussion: A very brief discussion of the results obtained on the usefulness of MLHPs in primary care was carried out. Authors should discuss and compare the results of the SRs evaluated with the available literature in the study topic. In the document, at times the discussion of the results is oriented to a context of low- and middle-income countries and at other times to the context of India. It would be helpful for authors to target their paper to LMIC with an emphasis on India at the discretion of the authors.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "6579", "date": "10 May 2021", "name": "Sandeep Moola", "role": "Author Response", "response": "Dear Guido, Thank you for taking out time to review the paper and for your valuable comments. We will revise and upload a new version of the article based on your feedback. Thanks again. Regards Sandeep" }, { "c_id": "6618", "date": "10 May 2021", "name": "Sandeep Moola", "role": "Author Response", "response": "Abstract (Introduction: Provide only necessary information to inform the context of the study. Methods: There is insufficient information about the study methods (quality of the included studies, stakeholder engagement, others). Results: no results are presented on the evaluation of the quality of the evidence. Conclusion: The conclusion does not emphasize that the evidence is of low or moderate quality on the usefulness of MLHPs in primary healthcare, and this should be mentioned). Thank you for your comments. We have revised the abstract incorporating the necessary changes (page 2). Introduction: The need for a rapid evidence synthesis for the study topic is not explicitly justified. I believe that the word \"gratis\" in the last paragraph is not suitable. The introduction is focused on India. This topic is suitable for LMIC. Hence, I recommend focusing the narrative of the manuscript on LMIC. We have now provided a statement to justify the conduct of a rapid evidence synthesis on the study topic. We have deleted the word “gratis” in the last paragraph. (page 3) We included some information relevant to LMICs to reflect the context and the nature of the findings of this review. Methods: Specify stakeholder participation. The methodology describes that a single reviewer performed the screening process (specify the reason for this). We included relevant information on stakeholder participation (page 4). Thank you for an excellent point on single reviewer screening. We used a single reviewer approach for study screening and selection based on the requirements of the review and resources available at the time. At the time, we considered this as a reasonable approach, as it involved using a single experienced reviewer (SM) for screening with a random verification of a subset of screening records by another experienced reviewer (SB). Results: Specify characteristics of the documents included in the analysis. There are tables that could be merged (table 2 to 6) to reduce the number of appendices since the last ones include data that should be presented in the manuscript body. Thank you. We briefly described the key characteristics of the documents under each domain of interest. We note your point about merging tables, but they have been kept separate for each domain of interest to avoid long tables and avoid confusion. We included tables related to essential characteristics of the SRs and the AMSTAR-2 checklist in the extended data file to limit the number of tables in the manuscript body. However, we did try to merge the tables, but the format appeared inappropriate. Discussion: A very brief discussion of the results obtained on the usefulness of MLHPs in primary care was carried out. Authors should discuss and compare the results of the SRs evaluated with the available literature in the study topic. In the document, at times the discussion of the results is oriented to a context of low- and middle-income countries and at other times to the context of India. It would be helpful for authors to target their paper to LMIC with an emphasis on India at the discretion of the authors. Thank you for your valuable comments on the discussion section. We have revised the discussion section in light of the feedback." } ] } ]	1	https://f1000research.com/articles/9-616
https://f1000research.com/articles/10-366/v1	10 May 21	{ "type": "Case Report", "title": "Case Report: Breast cancer-associated paraneoplastic stiff person syndrome: anastrozole monotherapy insufficient for symptom improvement", "authors": [ "Justin Cordova", "Blessie Nelson", "Ashley Brizendine", "David Pacheco", "Maurice Willis", "Avi Markowitz", "Justin Cordova", "Blessie Nelson", "Ashley Brizendine", "David Pacheco", "Maurice Willis" ], "abstract": "Stiff person syndrome (SPS) is a rare clinical disorder presenting with progressive muscle stiffness and painful spasms. Its ill-defined mechanism and variable presentation make diagnosis a challenge, though it is associated with a range of specific auto-antibodies. One particular antibody, anti-amphiphysin, is found in the presence of breast or lung malignancy and leads to a disorder termed paraneoplastic SPS (PSPS). Our patient, an 83-year-old woman, presented with bilateral leg weakness, spasms, and left clubfoot over a period of three months. She also reported a lump in her left breast for which she had not sought treatment over the past 10 years. Her ankle radiograph was negative for fractures and dislocations, while an MRI of the left leg was negative for plexopathies. Electromyography was suggestive of an SPS disorder and a positive anti-amphiphysin test indicated a diagnosis of PSPS. Her symptoms were managed with baclofen, diazepam, and five cycles of therapeutic plasma exchange (TPEX) over 10 days. Breast imaging revealed a 4.5-cm left breast lesion, later biopsy-confirmed as invasive ductal carcinoma (ER+, PR+, HER2−). The patient declined definitive surgical management, opting instead for once-daily anastrozole 1 mg as hormonal therapy. This regimen was not sufficient to lead to symptomatic improvement over a period of more than 30 days, and the patient expired less than 45 days after discharge. To our knowledge, this is the first case of PSPS to be treated in this manner. Our report illustrates that conservative management with anastrozole monotherapy was not sufficient to lead to symptomatic improvement in this form of paraneoplastic syndrome, suggesting the need for more aggressive pharmacological or definitive surgical intervention in order to produce symptom improvement and/or resolution.", "keywords": [ "Stiff person syndrome", "Paraneoplastic", "Invasive ductal carcinoma", "Anastrozole" ], "content": "Introduction\n\nStiff person syndrome (SPS) is part of an exceedingly rare spectrum of SPS disorders. Classic SPS presents with painful muscle spasms, increased sensitivity to stimuli, and gradual muscle stiffness, leading without intervention to spinal deformity and significant disability1,2. Though the pathophysiology is poorly understood, it is often accompanied by the presence of anti-glutamic acid decarboxylase antibodies or anti-glycine receptor antibodies. In rare cases of breast and lung malignancy, it can present with anti-amphiphysin antibodies and is termed paraneoplastic SPS (PSPS)3. These antibodies have also been associated with paraneoplastic cerebellar degeneration4 and a rare presentation of SPS with transverse myelitis5, both of which are beyond the scope of this current discussion. We present a case of PSPS in the setting of occult breast malignancy. Current management typically involves a three-pronged approach consisting of immunotherapy, symptomatic control, and tumor eradication1,3. Our case is unique in that our patient declined definitive surgical intervention and radiation, opting instead for conservative therapy with anastrozole. To our knowledge, this is the first such case to be treated in this manner. This article was previously presented as a virtual poster at the 2020 American College of Physicians Texas Chapter Meeting on November 7, 2020.\n\n\nCase presentation\n\nThe patient was an 83-year-old Caucasian woman who presented with bilateral leg weakness, painful leg spasms, and left talipes equinovarus (Figure 1). Her symptoms started three months prior to presentation, but worsened rapidly and severely limited her ambulation. She was a retired postmistress and was most alarmed by her reduced mobility, as she transitioned from walking unassisted to using a cane and eventually to a wheelchair over a period of two weeks. Her past medical history included celiac disease and an unspecified thyroid disorder, while a review of her family history revealed two sisters with prior episodes of breast cancer. On physical examination, her cranial nerve exam was normal, as was her motor exam in the bilateral upper extremities. On examination of her lower extremities, however, 3/5 strength was noted in her proximal left lower extremity with 4+/5 on the right. She also showed 3/5 strength in ankle dorsal and plantar flexion on the left side, along with absent ankle reflex and extensor plantar response. She reported a lump in her left breast, present for the past 10 years. Further workup included a left ankle radiograph, which was negative for fractures or dislocations (Figure 2), a spinal MRI without evidence of plexopathy, a negative test for ganglioside antibodies, and a negative myositis panel. Electromyography showed spontaneous ongoing muscle activity at rest, and co-activation of agonist/antagonist muscle groups in the bilateral lower extremities, indicating a central process like SPS. She was given diazepam and baclofen for symptom relief and, after confirmation of positive neuronal anti-amphiphysin antibodies in the cerebrospinal fluid, the patient underwent five cycles of therapeutic plasma exchange (TPEX) over a period of 10 days. She did not report any functional lower leg improvement during this time. Breast imaging identified a 4.5-cm left breast mass (Figure 3), with regional spread to the left axillary lymph nodes but without distant metastasis. Pathology confirmed invasive ductal carcinoma (ER+, PR+, HER2−) with a Ki-67 index of 50%, grade T2cN3aM0, clinical prognostic stage IIIB. The patient declined chemotherapy and surgical resection, agreeing instead to hormonal therapy in the form of once-daily anastrozole 1 mg. She passed away less than 45 days after discharge without showing significant improvement in symptoms of PSPS.\n\n\nDiscussion\n\nDespite the debilitating nature of PSPS, it is a clinically rare disorder without a clear delineation of optimal treatment practices. Disorders that fall upon the SPS spectrum have an estimated incidence of 1 case per million per year, but the paraneoplastic variant of the disease is particularly uncommon, comprising less than 10% of all SPS cases6. A review of current literature reveals a wide range of management options for PSPS, focusing primarily on treatment of the underlying malignancy and on symptomatic control. For muscle spasms and improvement in functional status, GABA agonists like diazepam or baclofen seem to be the preferred choice, though there is also a role for therapeutic plasma exchange and immunotherapy in refractory cases1.\n\nA myriad of approaches has been proposed for the treatment of underlying malignancy, based largely on the characteristics of the cancer itself and on the condition of the patient. The management options range from surgical intervention alone, as one case report detailed drastic symptomatic improvement after tumor resection in a woman with PSPS7, while many others included an aggressive chemotherapy regimen with or without radiation therapy8,9. One treatment plan coupled an intensive rehabilitation program with a regimen of chemotherapy, leading to significant symptomatic improvement in a matter of weeks10. The chemotherapeutic agents which appear to be favored are doxorubicin, docetaxel, and cyclophosphamide, though rituximab, carboplatin, and trastuzumab have also been reported8,10. The major takeaway from the literature is that all reports of symptomatic improvement have included aggressive management of the underlying malignancy, whether through surgical resection, chemotherapy, radiation, or a combination of the three. No previous cases have reported on the use of anastrozole monotherapy in treating breast cancer-associated PSPS, which led us to report our results.\n\n\nConclusions\n\nThe case we present here is unique in that it developed from occult malignancy, present for as many as 10 years, and that the patient elected to pursue hormonal treatment of her breast cancer rather than definitive surgical or chemotherapeutic intervention. She unfortunately did not show any measurable symptomatic improvement on anastrozole, which we feel indicates a need for a more aggressive treatment regimen or a more prolonged course of hormonal therapy. In this particular case, anastrozole alone was not sufficient to produce clinical improvement or symptomatic resolution. To our knowledge, this is the first such report of breast-cancer associated PSPS to be treated with anastrozole and our hope is that it will be the grounds for further exploration of management options for this rare disease process.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and/or clinical images was obtained from the patient.", "appendix": "References\n\nBaizabal-Carvallo JF, Jankovic J: Stiff-person syndrome: insights into a complex autoimmune disorder. J Neurol Neurosurg Psychiatry. 2015; 86(8): 840–8. PubMed Abstract \| Publisher Full Text\n\nMcKeon A, Robinson MT, McEvoy KM, et al.: Stiff-man syndrome and variants: clinical course, treatments, and outcomes. Arch Neurol. 2012; 69(2): 230–8. PubMed Abstract \| Publisher Full Text\n\nBalint B, Meinck HM: Pragmatic Treatment of Stiff Person Spectrum Disorders. Mov Disord Clin Pract. 2018; 5(4): 394–401. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGrativvol RS, Cavalcante WCP, Castro LHM, et al.: Updates in the Diagnosis and Treatment of Paraneoplastic Neurologic Syndromes. Curr Oncol Rep. 2018; 20(11): 92. PubMed Abstract \| Publisher Full Text\n\nFaissner S, Lukas C, Reinacher-Schick A, et al.: Amphiphysin-positive paraneoplastic myelitis and stiff-person syndrome. Neurol Neuroimmunol Neuroinflamm. 2016; 3(6): e285. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMurinson BB, Guarnaccia JB: Stiff-person syndrome with amphiphysin antibodies: distinctive features of a rare disease. Neurology. 2008; 71(24): 1955–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nManhalter N, Györfi O, Boros E, et al.: Case report of a woman with anti amphiphysin positive stiff person syndrome. Ideggyogy Sz. 2017; 70(5–6): 213–216. PubMed Abstract \| Publisher Full Text\n\nKelly PA, Kuberski C: Stiff person syndrome: a case report. Clin J Oncol Nurs. 2014; 18(4): 465–7. PubMed Abstract \| Publisher Full Text\n\nAgarwal PA, Ichaporia NR: Glutamic acid decarboxylase antibody-positive paraneoplastic stiff limb syndrome associated with carcinoma of the breast. Neurol India. 2010; 58(3): 449–51. PubMed Abstract\n\nSmith SR, Fu JB: Paraneoplastic stiff person syndrome: Inpatient rehabilitation outcomes of a rare disease from two cancer rehabilitation programmes. J Rehabil Med. 2016; 48(7): 639–42. PubMed Abstract \| Publisher Full Text" }	[ { "id": "87059", "date": "30 Jun 2022", "name": "John E. Greenlee", "expertise": [ "Reviewer Expertise Neuroimmunology and remote effects of cancer on the nervous system" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a report of an 83 year old woman who presented with a longstanding breast mass, found to be an invasive ductal carcinoma, and with stiff person syndrome (SPS) associated with anti-amphiphysin antibodies detected in CSF. The major point of the manuscript (after the patient had failed plasma exchange) was that treatment of the tumor with anastrozole failed to produce improvement in the patient’s SPS. The patient is an interesting one, and the manuscript is well-written and well-illustrated. One suspects that therapeutic options may have been limited by the patient’s willingness to undergo treatment. However, there are several points where the manuscript would be improved.\n\nThe fact that the patient’s SPS failed to improve on anastrozole is not surprising, and I am not sure that this point only – which is the main point of the manuscript – would be particularly helpful to the clinician treating such a patient.\n\nIt would be helpful to know why the patient died. Was it from her neoplasm, or from the SPS, or from some other cause?\n\nThe patient “declined chemotherapy”. Does this mean that the patient declined other antineoplastic chemotherapy, or immunotherapy, or both?\n\nSPS, including paraneoplastic SPS, is an immune-mediated disease. I suspect that relatively few neurologists would limit therapy to antineoplastic therapy only. Again, it would be worth knowing if the treatment approach used was because the patient refused other modalities such as IVIG, corticosteroids, or rituximab. Ideally, how do the authors think this patient should have been treated?\n\nThe discussion covers tumor treatment fairly well. More detailed information should be given concerning the role of immunotherapy.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-366
https://f1000research.com/articles/10-364/v1	10 May 21	{ "type": "Case Report", "title": "Case Report: Literature review of a Complicated Postpartum Pubic Symphysitis case", "authors": [ "Dhekra Toumi", "Korbi Asma", "Marwa Badr", "Farouk Ennaceur", "Boujemaa Touati", "Ahmed Hajji", "Raja Faleh", "Dhekra Toumi", "Marwa Badr", "Farouk Ennaceur", "Boujemaa Touati", "Ahmed Hajji", "Raja Faleh" ], "abstract": "Post-partum pubic symphysitis is an exceptional pathology. Its diagnosis is difficult, based on a high clinical semiological finesse complemented by imaging examinations, the most specific of which are CT and pelvic MRI. The earlier the diagnosis, the better the evolution. We report a case of pubic symphysitis occurring in the aftermath of a low-pathway delivery discovered in a septic shock table. Evolution was good under antibiotic and heparin therapy.", "keywords": [ "Post-partum", "pubic symphysitis", "case report." ], "content": "Introduction\n\nSeptic arthritis of the pubic symphysis is a rare infection, often occurring in particular patients including: athletes with chronic pubic inflammation, women in the postpartum period, drug addicts, patients following a surgical treatment for urinary incontinence or patients with pelvic cancer.1,2\n\nTo date, it is an often unknown infection because it is sometimes difficult to diagnose especially as the nosology remains unclear in the literature. Indeed, it must be suspected in the presence of pelvic girdle pain with fever and sometimes lameness or painful irradiation towards the lower limbs, but the symptomatology can be misleading.3\n\nThis clinical presentation helps guide additional studies that are not always specific with many differential diagnoses. CT and MRI are the two most sensitive exams.3,4\n\nAs for treatment, there are several therapeutic options whose pillar is antibiotic therapy.5,6\n\nWe present here a case of pubic symphysitis complicating the postpartum period.\n\n\nCase report\n\nThis was a 31-year-old female Caucasian patient with no relevant personal or family history who was re-admitted at day 25 postpartum for septic shock. The patient was primigravid, with no significant pathological history.\n\nThe pregnancy was normal. The patient had a low-birth-weight newborn baby of 3750 g. The delivery was without incident. The day after the delivery, the physical examination was strictly normal and the patient was discharged.\n\nAt day 15 postpartum, the patient described a gait lameness due to bilateral inguinal pain, in the context of unencrypted fever. The patient took this to be common postpartum joint pain and self-medicated with analgesics. However, the evolution was marked by the aggravation of functional impotence becoming disabling with the appearance of a fever which did not respond to medical treatment; thus, the patient consulted us.\n\nClinical examination at admission found a confused patient, febrile at 39°C, heart rate of 160 bpm, with blood pressure at 80/60 mmHg, hypogastric defense and sensitivity to palpation of the pubic symphysis and hip mobilization. The diagnosis of septic shock was retained. After conditioning, a pelvic ultrasound was performed showing multiple hyperechogenic heterogeneous compartmentalized collections occupying almost the entire pelvis without visualization of the uterus or ovaries.\n\nWe then performed an abdomino-pelvic CT that showed multiple compartmentalized collections, communicating with peripheral enhancement and water contents, distributed in the form of cubicles of variable size and shape occupying almost the entire perineum (intra-muscular), fusing within the root muscles of the lower limbs, coxo–femoral joints, rectus muscles and along the iliacus and psoas muscles. Calcifications were found within these collections (osteolysis). The most voluminous cubicle was located opposite the the 15-mm pubic symphysis. Osteolysis of the ilio- and ischio-pubic branches responsible for total disjunction with pubic diastasis. Floating, marginal and total thrombus in places at the level of the iliac bifurcation, the left common and external iliac veins, the right external iliac vein and bilateral common femoral veins (Figure 1).\n\nA few hours after admission, the patient’s condition quickly deteriorated with a severe collapse and recourse to noradrenaline. After conditioning, a laparotomy was made with exploration resulting from the collection of two liters of frank pus from Retzius’ space, with the presence of free bone fragments and a significant osteolysis of the pubic symphysis.\n\nThe initial investigations were bacteriological sampling, removal of the bone sequester with slight bone and excision of the false membranes, an access to the cubicles at the perineal muscles and iliopsosas and cleaning with physiological serum. Then we entered into exploration of the peritoneal cavity, which was without anomalies. At the end of the operation, two undulating blades were installed at the root of the lower limbs and a Salem probe between the bladder and the uterus (Figure 2).\n\nBiologically there was moderate leukocytosis at 10,000 cells/mm3, associated with an inflammatory syndrome (CRP = 30 mg/L).\n\nThe diagnosis of septic arthritis of the complicated pubis of a state of septic shock with abscesses in adjacent muscles and extensive iliac venous thrombosis was therefore retained. The patient was placed on broad-spectrum probabilistic antibiotic therapy using vancomycin (500 mg four times per day) and Tienam (1 g four times per day) parenteral antibiotics and on a curative dose low-molecular-weight heparin. The bacteriological sampling done per-operatively and the blood cultures came back negative. The evolution was marked by progressive clinical improvement.\n\nA control CT was performed on day 13 of the treatment objectifying: 1) a right retroperitoneal collection of 5×7 cm; 2) a partial regression of the collections of the left iliopsoas muscle; 3) a raised wall delimiting multiple communicating cubicles; 4) a near total regression of the perineal collections, the right abdominal muscle and the roots of the lower limbs; 5) a stable aspect of venous thrombosis; and 6) as well as bilateral anterior osteolysis of the ilio and ischio pubic branches. Following these CT findings, a CT-guided puncture was then used in the retroperitoneal collection bringing back a thick greenish liquid with the installation of a drain (Figure 3).\n\nDual therapy was maintained for 6 weeks with an apyrexia as of day 3. Biological inflammatory syndrome disappeared after one month. The removal of the two initially placed corrugated blades was done after 3 weeks.\n\nThe patient was asymptomatic during the last check-up with a recoil of 8 months.\n\n\nDiscussion\n\nPubic symphysis represents less than 1% of all hematogenous osteomyelitis.7 They are exceptional in post-partum women.6 This is the first case encountered in our service. Pubic symphysis often occurs in a particular field causing symphysis lesions or locoregional infection.3\n\nIndeed, in post-partum women, in addition to the risk of lesions of the symphysis in peripartum (even in the absence of traumatic childbirth), the immunodeficiency due to pregnancy and delivery promotes the pillulation of germs. Concerning the pathophysiology, it is still poorly elucidated by Mynors in 1974 suggested that it is mainly pubic thrombosis due to the dramatic response to heparinization of resistant cases of pubic osteitis.8\n\nThe pubic symphysitis in post-partum is difficult and often late diagnosis because pain is often related to the circumstances of childbirth with recourse to self medication that will alleviate the symptomatology. According to a literature review of 100 cases of pubic symphysitis, published by Ross et al.6 In 2003, most patients reported pubic pain (68%). Pain is also localized to the groin (41%), thigh (15%) and hip (12%), probably due to radiation along the hip adductors, which insert onto the pubic symphysis.\n\nReaching the hip adductors also explains the pain during walking (59%) and hip movement (45%). Long delays between symptom onset and diagnosis were typical (mean, 29 days), due to insidious presentation and low clinical suspicion of this rare disease.6 In our patient the diagnosis was made 10 days after the onset of symptoms.\n\nIndeed, the secondary appearance of a pain that did not exist in early post-partum, its increasing intensity, or its unfavourable and unconventional evolution, the presence of a fever or a functional impotence associated especially after non-dystocic delivery are elements that can guide the diagnosis.9\n\nTo support the diagnosis or guide the samples two essential examinations to be requested are CT and MRI of the pelvis.3,9\n\nThese examinations typically show bone erosions, a bank abscess, enlargement or effusion of the pubic symphysis. MRI provides early detection of edema and inflammation of the bone and muscles, intra-articular effusion or abscess.4\n\nIt will then be necessary to search for the responsible germ. Staphylococci aureus (34%) and Pseudomonas aeruginosa (24%) are the bacteria most often incriminated.3 Pubic symphysis with group B streptococcus are exceptional.6 This bacteria can be isolated in blood cultures or post-partum lochia. If this investigation is negative, a local biopsy sample by trocar or surgical biopsy may be proposed. The low virulence of these bacteria, their long culture time, or prior antibiotic therapy may lead to the culture being wrongly considered sterile3 as is the case with our observation.\n\nIn this pathology, the biological signs are neither specific nor constant. In the majority of cases, there is an inflammatory syndrome with increased sedimentation rate, but leukocytosis is inconstant, exceeding 11 000 cells per mm3 in only 35% of cases according to the same series.6 For our case, the inflammatory syndrome is moderate with leukocytosis at 10000 and CRP at 30 despite the severity of the clinical picture and per-operative findings.\n\nThe treatment is based, classically, on antibiotic therapy of at least 6 weeks sometimes associated with the drainage of a collection.6 But there is no clear recommendation in the literature about which molecule to use.\n\nThe evolution is generally favorable in case of early diagnosis and treatment; however, 55% of patients required surgery either for debridement or for abscess evacuation.10 In the absence of treatment, the progression can be towards chronicity with the possibility of appearance of fistulas, bone sequesters and the possibility of pelvic cellulitis.11\n\nFor our patient, although she presented with septic shock and the per-operative findings were serious, the evolution was still favorable.\n\nThe control check is based mainly on the clinical evolution and the surveillance of the inflammatory syndrome because the imaging takes a long time to show a healing para port to the clinical cure. But for our patient, we asked early for a control scan of extensive associated lesions (venous thrombosis and abscess of the soft parts).\n\n\nConclusion\n\nPost-partum pubic symphysitis is a very rare condition with a difficult diagnosis. Two essential tests that should not be delayed to confirm the diagnosis: CT and pelvic MRI are required to confirm the diagnosis and eliminate complications. A treatment based on antibiotic therapy is to start from the diagnosis because the earlier treatment, the more favorable the evolution.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nWritten informed consent for publication of their clinical details and/or clinical images was obtained from the patient.", "appendix": "References\n\nCharles P, et al.: Arthrites septiques spontanées à streptocoques de la symphyse pubienne. La Revue de médecine interne. 2011; 32(7): e88–e90. Publisher Full Text\n\nRosenthal RE, Anderson Spickard W, Douglass Markham R, et al.: Osteomyelitis of the symphysis pubis: a separate disease from osteitis pubis. J Bone Joint Surg. 1982; 64: 123–8. PubMed Abstract\n\nSalomon S, Lasselin-Boyard P, Lasselin J, et al.: Symphysite pubienne infectieuse post-chirurgicale. Progrès en urologie. 2015; 25(3): 169–74. Publisher Full Text\n\nJarlaud T, Railhac JJ, Sans N, et al.: Normal and patho-logic symphysis pubis: value of imaging. J Radiol. 2001; 82: 425–36. PubMed Abstract\n\nGrace JN, Sim FH, Shives TC, et al.: Wedge resection of the symphysis pubis for the treatment of osteitis pubis. J Bone Joint Surg. 1989; 71: 3586–94. PubMed Abstract\n\nRoss JJ, Hu LT: Septic arthritis of the pubic symphysis: review of 100 cases. Medicine. 2003; 82(5): 340–5. PubMed Abstract \| Publisher Full Text\n\nArlet J, Bouteiller G, Durroux R, et al.: Ostéites pubiennes et ischio-pubiennes. Étude bactériologique et histopathologique de l’os pubien. Rev Rhum. 1981; 48: 101—6.\n\nMynors JM: Osteitis pubis. J Urol. 1974 Nov; 112(5): 664–5.\n\nKehila M, Majdoub M, Zegha D: La symphysite pubienne du post-partum: un diagnostic difficile. Pan Afr Med J. 2014, vol. 16, no 1. PubMed Abstract \| Publisher Full Text Publisher Full Text\n\nDucrotoy V, Fournet P, Vittecoq O, et al.: Postpartum septic arthritis - Two case reports. J Gynecol Obstet Biol Reprod (Paris). 1998 Jun; 27(4): 449–54. PubMed Abstract\n\nChoi H, McCartney M, Best TM: Treatment of osteitis pubis and osteomyelitis of the pubic symphysis in athletes: a systematic review. Br J Sports Med. 2011 Jan; 45(1): 57–64. PubMed Abstract \| Publisher Full Text Publisher Full Text" }	[ { "id": "85104", "date": "01 Jun 2021", "name": "Filippo Maselli", "expertise": [ "Reviewer Expertise muscoloskeletal disorders", "differential diagnosis", "screening for referral", "low back pain", "sport physical therapy", "spinal manipulation", "running related injuries" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI realize that authors have many journals to consider when they want to publish their work, so I appreciate their interest in F1000Research; I am very sorry not to be able to write in a more positive way. It is evident that the authors have put a great deal of effort into this project and I want to praise their efforts, but unfortunately, the actual contribution from their paper to orthopaedic surgery or physical therapy practice' literature is not clear or strong. The manuscript as currently written suggests that it might be suitable for sharing information about this case, but the case report they reported is not representative to state with certainty the conclusions. I am very happy to say that I can review this paper after major revision. It may be that the authors would like to consider resubmitting it, in which case I hope that the comments from my review may help them to revise it before resubmitting it. These comments are given below.\nFor a case report, a lot of information about the case is missing; there is no information which could be an aid in the clinical practice of health professionals who may encounter similar cases. More in depth, the background and clinical reasoning on this patient's clinical presentation is very poor.\n\nThe case presentation section is missing more data about patient: history of smoking, sport activity, work activity, etc.\n\nNeither other differential diagnosis hypothesis was supposed, why?\n\nFigures of diagnostic bioimages are missing details and I suggest to add arrows in the figures for marking the clinical findings; they would have helped to better understand the clinical condition and treatment.\n\nWhat are the real differences compared to similar cases already present in the literature? What really is the patient's clinical condition, Pubic Symphysitis, caused by? Was an underlying medical condition investigated that could support Pubic Symphysitis?\n\nI suggest to add to the references' list, to aid sustaining the background and clinical reasoning in the case presentation section, the following:\nFinucane et al. (20201).\n\nGalliker et al. (20202).\n\nMaselli et al. (20203).\n\nI am very sorry not to be able to write in a more positive way but I should like to thank you for give me an opportunity to consider this interesting work for indexing.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] }, { "id": "96788", "date": "21 Oct 2021", "name": "Dulce A. Oliveira", "expertise": [ "Reviewer Expertise Biomedical engineering", "modelling childbirth", "finite element analysis" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMy area of expertise is biomedical engineering so reviewing this paper is quite difficult.\nThe authors show that it is a case that deserves to be studied because of its rareness, but the case they present should be explored more so that it can also be useful for other colleagues. One would expect more information about the patient, more detail about the choices made and why, alternatives that could have been pursued and justification for not doing so, etc.\nThe introduction is a bit poor, it would be interesting to present some numbers, for example, the percentage of women who suffer from this infection, the percentage of women who have their postpartum aggravated due to this condition, etc. The paragraph regarding treatments could also be further explored, even though the treatment mentioned was the one used in this case report.\nIn the introduction you refer to \"Septic arthritis of the pubic symphysis\". Does this correspond to pubic symphysitis?\nWhat is the main goal of the study? Will the present study help on the early diagnosis of future pubic symphysitis cases?\nIn discussion section you state: “Pubic symphysis represents less than 1%”, shouldn’t it be pubic symphysitis? Still in the discussion section you mention “The diagnosis of septic shock was retained.”. What led to this diagnosis? Were other possible conditions considered?\nRegarding Figure 1, and to help readers, the zones mentioned should be highlighted in the figure. The same for Figure 3, include a more detailed description in the figure to help the reader to follow the concepts.\n“leukocytosis at 10000 and CRP at 30”, please indicate the units.\nThe discussion could be more focused on the patient, on what could have been done to avoid such an aggravated condition and suggestions for other patients to prevent this complication.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-364
https://f1000research.com/articles/10-361/v1	07 May 21	{ "type": "Case Report", "title": "Case Report: Guitarist’s cramp as the initial manifestation of dopa-responsive dystonia with a novel heterozygous GCH1 mutation", "authors": [ "Takafumi Hasegawa", "Tatsuhiko Hosaka", "Ryuhei Harada", "Ichiro Kawahata", "Kyoko Hoshino", "Naoto Sugeno", "Akio Kikuchi", "Masashi Aoki", "Tatsuhiko Hosaka", "Ryuhei Harada", "Ichiro Kawahata", "Kyoko Hoshino", "Naoto Sugeno", "Akio Kikuchi", "Masashi Aoki" ], "abstract": "Dopa-responsive dystonia (DRD), also known as Segawa syndrome, is a phenotypically and genetically heterogeneous group of neurological disorders that typically presents as early-onset lower limb dystonia with diurnal fluctuation, and exhibits a marked, persistent response to levodopa. Heterozygous loss-of-function mutations in the guanosine triphosphate cyclohydrolase 1 (GCH1) are the most common cause of DRD. In addition to the classic form of the disease, there have been a number of studies addressing atypical clinical features of GCH1 related DRD with variable age of onset. This report describes a 37-year-old Japanese male patient with a 10-year history of focal upper limb dystonia that initially emerged as task-specific, guitarist’s cramp. The dystonic symptoms responded very well to levodopa treatment, and genetic analysis identified a novel heterozygous mutation in the C-terminal catalytic domain of GCH1. Insufficient recognition of this treatable condition often leads to misdiagnosis, which causes delays in the patient receiving adequate dopamine replenishing therapy. A diagnostic trial with levodopa should be considered in all patients with relatively young-onset dystonia, whether they have classic features of DRD or not.", "keywords": [ "Guitarist’s cramp", "dystonia", "task-specific", "dopa-responsive", "dopamine", "Segawa syndrome", "GCH1", "DYT5a" ], "content": "Introduction\n\nDopa-responsive dystonia (DRD, Segawa syndrome) is a rare movement disorder typically characterized by childhood-onset walking difficulties due to lower limb dystonia, diurnal fluctuation, and dramatic, sustained response to relatively low-dose levodopa treatment1. Striatal dopamine deficiency due to loss-of-function mutations in the guanosine triphosphate cyclohydrolase 1 (GCH1, EC 3.5.4.16) is the most common etiology in the autosomal dominant form of DRD (DYT5a, OMIM 128230), in which incomplete penetrance and variable phenotype are observed2. Owing to its diverse clinical presentations and poor recognition by general practitioners, DRD is under-reported and may be mistaken for other conditions such as cerebral palsy or psychogenic movement disorder. In this report, an unusual, adult case of DRD/DYT5a initially presenting as guitarist’s dystonia is described.\n\n\nCase report\n\nThe 37-year-old, right-handed Japanese man with a professional career as a guitar player, was referred to our hospital for the reassessment of upper limb dystonia. Upon assessment, no family history of neurological disorders was reported, and he did not use any regular medication before onset. At the age of 27 years, he began to spend most of his time practicing guitar and three months later, he felt difficulty in picking a pick due to excessive wrist extension and intermittent, tremulous finger movement in the right hand. At the age of 29 years, he was diagnosed with having guitarist’s cramp, for which the oral administration of clonazepam (1.5 mg/day) and trihexyphenidyl (6 mg/day) were prescribed. This treatment proved to be ineffective. After switching from guitar to piano, these strange movements transiently disappeared but later reappeared. Meanwhile, the task-specificity of hand dystonia was gradually lost, and the disabling hand dystonia was induced by other daily activities including opening/closing a screw cap bottle and the brushing of his teeth. After five years from the onset, he had considerable difficulty in playing instruments, and finally, he decided to end his musical activities.\n\nDuring consultation, neurological examination of cranial nerves, motor function, coordination, sensory function, and autonomic function showed unremarkable results. This was with the exception of action-induced dystonic posturing of the right upper limb with excessive wrist extension and hyperextension of the fingers, though these findings only became prominent during voluntary, skilled movement (Video S1, Extended data3). Neither sensory trick nor diurnal variation was observed. Other abnormalities, including cognitive dysfunction, parkinsonism, pyramidal signs, and cerebellar ataxia, were not detected. Workup including electrolytes, renal function tests, complete blood count, liver function tests and urinalysis were unremarkable. In addition, the levels of serum copper and ceruloplasmin were normal. Cranial magnetic resonance imaging and dopamine transporter (DAT) imaging with 123I-β-CIT (2β-carbomethoxy-3β-(4-iodophenyl) tropane) single-photon emission computed tomography were normal (Figure 1). The diagnosis of DRD was suspected since the oral administration of levodopa (300 mg per day) showed dramatic, sustained improvement of the dystonic symptoms on the following day (Video S2, Extended data3), and cerebrospinal fluid analyses revealed a significant decrease in homovanillic acid (22.6 ng/ml, normal range: 41.6–178 ng/ml), 5-hydroxyindoleacetic acid (9.1 ng/ml, normal range: 20.0–96.0 ng/ml), and total neopterin (2.0 pmol/ml, normal range: 9.0–20.0 pmol/ml) levels4. The clinical suspicion of DRD was further strengthened by exome analysis and Sanger sequencing showing a novel heterozygous mutation c. 542T>G (p. Val181Gly) in the first amino acid of exon 5 in GCH1 gene (Figure 2A). The first Val in the 5th exon was located in the enzymatic core of GCH1 at the C-terminus5, and is highly conserved across species (Figure 2B). This amino acid substitution was predicted to be pathogenic using the in silico analysis tools, SIFT and PolyPhen-2. In the half-year follow-up, the patient’s dystonic symptom was well-controlled by the levodopa therapy without any adverse side effects.\n\nA: Cranial magnetic resonance imaging shows no abnormality in (a) T1 and (b) T2 axial sequences. B: Transverse dopamine transporter image with 123I-β-CIT (2β-carbomethoxy-3β-(4-iodophenyl)tropane) single-photon emission computed tomography demonstrates normal binding of radioligand in both caudate nuclei and putamina.\n\nA: Sanger sequencing chromatogram which shows a novel heterozygous mutation c. 542T>G (p. Val181Gly) in the first amino acid of exon 5 in the GCH1 gene (black arrow). B: Amino acid sequence alignment of the GCH1. Note that the first Val in exon 5 (highlighted in red) is highly conserved across species. Residues involved in catalysis are highlighted with black triangles\n\n\nDiscussion\n\nIn most cases of adult-onset focal limb dystonia, the exact, underlying etiology remains unclear, but in some cases, dystonia occurs due to specific biochemical defects and genetic alterations6. A good example is the DRD caused by genetic defects in dopamine biosynthesis. Deficiency of GCH1, a rate-limiting enzyme in the biosynthetic pathway of tetrahydrobiopterin, is the most common and well-characterized condition that manifests as DRD7. In contrast to the childhood-onset GCH1-related DRD, adult cases often present parkinsonism followed by dystonia, and the movement problems progress slowly without diurnal variation8,9. Furthermore, a number of studies describing atypical or incompatible features of GCH1 deficient-DRD with variable age of onset exist2. Theoretically, DRD is considered to be a neurotransmitter disorder that is not accompanied by the nigrostriatal degeneration; however, some patients with adult-onset dystonia-parkinsonism or Parkinson’s disease without any dystonic feature carrying GCH1 mutation have been known to show abnormal DAT imaging2.\n\nThe most conspicuous feature in this patient’s case is that the dystonic movement which started to present as a guitarist’s cramp. Although infrequent, a few reports of DRD/DYT5a presenting task-specific dystonia in the upper limb have been presented10,11. Among them, the most common phenotype was writer’s cramp, which became obvious during the disease progression. Conversely, there was only one case report which presented adult-onset guitarist’s cramp in the family carrying GCH1 truncating mutation (p. Arg216stop)12. A genotype-phenotype correlation was unlikely, because even among members of the same family with the same mutation, the movement symptoms were different. While the pathophysiology of task-specific or occupational dystonia still remains elusive, overtraining and genetic predisposition may contribute to the expression of dystonic symptoms. Indeed, about 20% of patients with musician’s dystonia have a family history of the disorder, and a genome-wide association study demonstrated a possible link between musician’s dystonia and the intronic variant in the arylsulfatase G gene13. Furthermore, patients with DYT1 and DYT11 dystonia rarely have writer’s cramp as the cardinal symptom14,15. Although we did not conduct personality and behavioral assessment of the patient, specific personality traits including susceptibility to anxiety and stress, or perfectionism may also increase the risk of developing dystonia16.\n\nIn conclusion, we reported a case of a 37-year-old male who was diagnosed with DRD/DYT5a, which was confirmed through genetic sequencing. The patient presented focal upper limb dystonia which first emerged as task-specific, guitarist’s cramp. The clinical heterogeneity of DRD often makes diagnosis difficult and leads to therapeutic delay. Our experience further underscores the broad clinical presentations of DRD as well as advocating for the diagnostic value of trying levodopa and genetic testing in dystonia.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nDryad: Dopa-responsive dystonia patient response before and after levodopa treatment. https://doi.org/10.5061/dryad.pzgmsbckd3.\n\nThis project contains the following extended data:\n\n- Video S1 (.MOV video of the neurological assessment on admission. Dystonic posturing of the right upper limb with excessive wrist extension and hyperextension of the fingers are seen during the opening of a screw cap bottle. No parkinsonism, pyramidal signs or cerebellar ataxia are observed)\n\n- Video S2 (.MOV video taken on the following day after the oral administration of 300 mg per day of levodopa. The task-specific dystonia in the right upper limb is dramatically improved).\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\n\nConsent\n\nWritten informed consent for publication of the clinical details, diagnostic images and videos was obtained from the patient.", "appendix": "Author contributions\n\n\n\nConceptualization: THA\n\nFormal Analysis: THA\n\nFunding Acquisition: THA, IK\n\nInvestigation: THA, THO, RH, IK, NS, AK\n\nMethodology: THA, IK\n\nProject Administration: THA\n\nResources: THA, THO, RH, NS, AK,\n\nSupervision: KH, MA\n\nValidation: THA\n\nVisualization: THA\n\nWriting – Original Draft Preparation: THA\n\n\nReferences\n\nSegawa M, Hosaka A, Miyagawa F, et al.: Hereditary progressive dystonia with marked diurnal fluctuation. Adv Neurol. 1976; 14: 215–233. PubMed Abstract\n\nWijemanne S, Jankovic J: Dopa-responsive dystonia--clinical and genetic heterogeneity. Nat Rev Neurol. 2015; 11(7): 414–424. PubMed Abstract \| Publisher Full Text\n\nHasegawa T: Dopa-responsive dystonia patient response before and after levodopa treatment. Dryad. Dataset, 2021. http://www.doi.org/10.5061/dryad.pzgmsbckd\n\nBlau N, Bonafé L, Thöny B: Tetrahydrobiopterin deficiencies without hyperphenylalaninemia: diagnosis and genetics of dopa-responsive dystonia and sepiapterin reductase deficiency. Mol Genet Metab. 2001; 74(1–2): 172–185. PubMed Abstract \| Publisher Full Text\n\nMaita N, Hatakeyama K, Okada K, et al.: Structural basis of biopterin-induced inhibition of GTP cyclohydrolase I by GFRP, its feedback regulatory protein. J Biol Chem. 2004; 279(49): 51534–51540. PubMed Abstract \| Publisher Full Text\n\nAlbanese A, Di Giovanni M, Lalli S: Dystonia: diagnosis and management. Eur J Neurol. 2019; 26(1): 5–17. PubMed Abstract \| Publisher Full Text\n\nIchinose H, Ohye T, Takahashi E, et al.: Hereditary progressive dystonia with marked diurnal fluctuation caused by mutations in the GTP cyclohydrolase I gene. Nat Genet. 1994; 8(3): 236–242. PubMed Abstract \| Publisher Full Text\n\nKikuchi A, Takeda A, Fujihara K, et al.: Arg(184)His mutant GTP cyclohydrolase I, causing recessive hyperphenylalaninemia, is responsible for dopa-responsive dystonia with parkinsonism: a case report. Mov Disord. 2004; 19(5): 590–593. PubMed Abstract \| Publisher Full Text\n\nWeng YC, Wang CC, Wu YR: Atypical presentation of dopa-responsive dystonia in Taiwan. Brain Behav. 2018; 8(2): e00906. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTrender-Gerhard I, Sweeney MG, Schwingenschuh P, et al.: Autosomal-dominant GTPCH1-deficient DRD: clinical characteristics and long-term outcome of 34 patients. J Neurol Neurosurg Psychiatry. 2009; 80(8): 839–845. PubMed Abstract \| Publisher Full Text\n\nTassin J, Dürr A, Bonnet AM, et al.: Levodopa-responsive dystonia. GTP cyclohydrolase I or parkin mutations? Brain. 2000; 123(Pt 6): 1112–1121. PubMed Abstract \| Publisher Full Text\n\nBandmann O, Valente EM, Holmans P, et al.: Dopa-responsive dystonia: a clinical and molecular genetic study. Ann Neurol. 1998; 44(4): 649–656. PubMed Abstract \| Publisher Full Text\n\nLohmann K, Schmidt A, Schillert A, et al.: Genome-wide association study in musician's dystonia: a risk variant at the arylsulfatase G locus? Mov Disord. 2014; 29(7): 921–927. PubMed Abstract \| Publisher Full Text\n\nGerrits MCF, Foncke EMJ, Koelman JHTM, et al.: Pediatric writer's cramp in myoclonus-dystonia: maternal imprinting hides positive family history. Eur J Paediatr Neurol. 2009; 13(2): 178–180. PubMed Abstract \| Publisher Full Text\n\nGasser T: Inherited myoclonus-dystonia syndrome. Adv Neurol. 1998; 78: 325–334. PubMed Abstract\n\nTomić A, Petrović I, Pešić D, et al.: Is there a specific psychiatric background or personality profile in functional dystonia? J Psychosom Res. 2017; 97: 58–62. PubMed Abstract \| Publisher Full Text" }	[ { "id": "84972", "date": "08 Jun 2021", "name": "Hiroshi Ichinose", "expertise": [ "Reviewer Expertise Biochemistry" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors described an adult male case of DRD/DYT5a who initially developed focal upper limb dystonia and diagnosed as guitarist’s dystonia, and found that the dystonic symptoms responded very well to levodopa treatment. They propose that a diagnostic trial with levodopa should be considered in all patients with relatively young-onset dystonia regardless of whether they have typical features of DRD or not. This paper is worthwhile publishing to report that adult-onset focal limb dystonia can be presented in patients with DRD/DYT5a, suggesting a broad clinical presentation of DRD/DYT5a.\nThe reviewer would like to ask for the following minor revision to the manuscript;\nThe authors should discuss the benefits and possible adverse effects of levodopa treatment for the patients with dystonia, whereas the authors propose the diagnostic value of levodopa treatment.\n\nThe biochemical analysis of HVA and neopterin in the CSF is beneficial for a differential diagnosis of DRD/DYT5a from other types of dystonia/parkinsonism. It should be discussed clearly in the text.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "90523", "date": "09 Aug 2021", "name": "Taku Hatano", "expertise": [ "Reviewer Expertise Parkinson's disease", "Movement disorders" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nHasegawa and colleagues reported the case with DYT5, who exhibited the focal dystonia mimicking musician's cramp. They described the patient’s medical story and assessed the patient’s state by appropriate examinations, including neurological examination, cranial MRI, DAT scan, and genetic analysis. They also mentioned the important learning points. Thus, I am interested in this paper. But I have a comment on this paper.\nMajor point:\nThe authors pronounced that in the early stage, the patient exhibited the mimicking guitarist’s cramp, but his dystonia emerged in not only task-specific but also usual situations. This finding is different from idiopathic task-specific dystonia in the disease course. The authors should discuss the difference between genetic-related and idiopathic task-specific dystonia.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "90521", "date": "17 Aug 2021", "name": "Juei-Jueng Lin", "expertise": [ "Reviewer Expertise Parkinson's disease and related disorders" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nHasegawa T et al. report a young adult man with a 10-year history of upper limb dystonia that mimicked as task-specific dystonia. His dystonia responded well to Levodopa treatment and his genetic study revealed a novel heterozygous mutation of GCH1 gene. Therefore, they conclude task-specific dystonia can also be one phenotype of Segawa syndrome or dopa-responsive dystonia (DRD). Meanwhile, a series of neuroimaging studies of this case including CT, MRI and SPECT of the brain were all negative results. I am interested in this case, but I have three major comments on this paper:\nThe authors emphasized that the patient presented guitarist’s cramp in his early stage of disease, three months later after an intensive practice of guitar. Meanwhile, they also described the dystonia can also appear in playing other instruments and in some daily activity. This manifestation is different from the disease course of idiopathic task-specific dystonia. The authors should discuss the differences between idiopathic task-specific dystonia and genetic related task-specific dystonia.\n\nThe patient responded well to treatment with levodopa and was subsequently diagnosed to be DRD, but did not have a family history. The genetic testing for GCH1 gene revealed a novel heterozygous mutation of c. 542T>G. However, the paper does not mention genetic analysis for GCH1 of his parent or siblings. Therefore, the authors should provide the genetic testing of GCH1 of other families to confirm genetic basis of their reported patient, In addition, they also can discuss the intrafamilial variability of photype in DRD family.\n\nThe authors also reported a normal presynaptic nigrostriatal dopamine function by a normal SPECT study in their DRD case. There have been reports in literature with an abnormal presynaptic nigrostriatal dopamine function in DRD patient presented with either dystonia or Parkinsonism. The author should discuss the relationship of presynaptic dopamine function in patients with DRD presented with variably clinical phenotypes.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-361
https://f1000research.com/articles/10-359/v1	07 May 21	{ "type": "Research Article", "title": "Cost analysis of outpatient services for major external structural birth defects: An ingredient approach in selected hospitals in Kiambu County, Kenya", "authors": [ "George N. Agot", "Joseph K. Wang'ombe", "Marshal M. Mweu", "Joseph K. Wang'ombe", "Marshal M. Mweu" ], "abstract": "Background: Major external structural birth defects are known to exert an enormous economic burden on individuals and health services; however, they have been vastly unappreciated and underprioritized as a public health problem in settings where cost analyses are limited. Objective: The objective of this study was to conduct a cost analysis of outpatient services for major external structural birth defects in selected hospitals in Kiambu County, Kenya. Methods: A hospital-based cross-sectional study design was adopted in four hospitals where an ingredient approach was used to retrospectively gather data on cost drivers for interventions consisting of castings, bracings, and tendonectomies for the under-fives from health care providers’ perspectives for a one-year time horizon (January 1st, 2018, to December 31st, 2018). The hospitals were selected for providing outpatient corrective and rehabilitative services to the under-fives. Prevalence-based morbidity data were extracted from outpatient occupational therapy clinic registers, whereas staff-time for the hospitals’ executives comprising the medical superintendents, chief nursing officers, orthopedic surgeons, and health administrative officers were gathered through face-to-face enquires from the occupational therapists being the closest proxies for the officers. Following a predefined inclusion criterion, 349 cases were determined, and associated cost drivers identified, measured, and valued (quantified) using prevailing market prices. The costs were categorized as recurrent, and unit economic costs calculated as average costs, expressed in U.S Dollars, and inflated to the U.S Dollar Consumer Price Index from January 2018 to December 2018. Results: The unit economic cost of all the cases was estimated at $1,139.73; and $1,143.51 for neural tube defects, $1,143.05 for congenital talipes equinovarus, and $1,109.81 for congenital pes planus. Conclusions: The highest economic burden of major external structural birth defects in the county was associated with neural tube defects, followed by congenital pes planus despite having the fewest caseloads.", "keywords": [ "Major external structural birth defects", "costing analysis", "outpatient services", "ingredient approach", "Kenya" ], "content": "Introduction\n\nMajor external structural birth defects (MESBDs) are defined as physical abnormalities of intrauterine origin present from birth, detectable visually, and having significant health and development impacts1–4. These defects are potentially fatal and those children who survive beyond infancy require substantial economic resources to deal with lifelong disabilities5–9. Worldwide, approximately 134 million births reportedly occur yearly, of which 7.9 million (6%) are born with at least a major birth defect, mostly affecting the central and musculoskeletal systems1–4,10. Although about 3.3 million of these children die before they are five years old, the 3.2 million who survive may be disabled for life if sufficient resources are not dedicated to corrective and rehabilitative health services1,2,8.\n\nMESBDs continue to occur exerting an enormous economic burden on individuals and health services in developing countries; however, they have been vastly ignored and unappreciated as public health problems due to limited estimation of the associated costs attributed to the scantiness of, and inaccurately profiled data on cost drivers1,2,9,11. Even though these defects remain a “silent” global public health problem, the highest-burden is shouldered by developing countries due to high prevalence of modifiable risk factors coupled with deficient expertise in economic evaluation studies1–4,6,8,9,12–15. Hospital charges for new-born children born with some forms of birth defect have been reported as four to eight times higher than those without any form of the defects16. Cost analysis is a partial economic method of evaluating health care programs used to compare the costs of at least two alternative interventions; however, cost studies are still useful even in the absence of comparative interventions as they can establish baseline economic costs of a health intervention17–19.\n\nChildren surviving beyond infancy could require restorative health services to reduce the adverse impacts associated with MESBDs1,2. These interventions are described as corrective and rehabilitative outpatient services consisting of castings, bracings, and tendonectomies whose monetary value is referred to as economic or opportunity costs17,18,20–24. The resources used in the provision of such services could be quantified through micro-costing (bottom-up) using an ingredient approach to gather data on the cost drivers by a step-down full costing technique, activity-based costing, time and motion, surveys, and manager interview techniques17,18,20,25–27. Alternatively, these inputs may be quantified by gross costing (top-down) using historical input outlay17,18,20,25–27.\n\nThe range, contexts, and extents of cost elements are determined by economic viewpoints consisting of health care providers’, individuals’, or societal perspectives that are informed by policy decisions known as the study objectives, and/or questions17,18,20,26. The existing market prices and opportunity costs (forgone benefits) are used to value the inputs in the monetary units categorized as recurrent and capital costs17,18,20–24. These costs are assigned to direct, indirect, and intermediate cost centers using a step-down accounting technique17,18,20–24. The costs of the inputs are sometimes shared among the cost centers thus referred to as overhead (shared or joint) costs which are proportionally allocated to the respective cost centers for estimating final economic costs17,18. Capital costs determined for more than a one-year time horizon should be considered for the differential-time discounting unlike recurrent costs17,18,20–24. Similarly, statistical and/or sensitivity analysis should be conducted to ascertain the robustness of the evaluation findings because of potential uncertainties arising from sample size determination and data collection methods for the cost drivers17.\n\nThe advancements in medical and surgical interventions are known to reduce the severity of lifelong physical disabilities related to MESBDs; however, their costs are catastrophic and prohibitive to many households and public health care systems globally1,2,9,15. Even though substantial resources are usually allocated to health care systems for the provision of corrective and rehabilitative health services for MESBDs, their costs are seldom estimated due to the rarity and stochasticity of the defects, scantiness of the cost data, inaccurately profiled cost information and inadequate costing expertise in developing countries19,20,28,29. The scarcity of local epidemiological data and differences in the epidemiological study design (prevalence/incidence-based) also impede the accuracy of the profiled cost data in developing countries19,20,28,29.\n\nEconomic costs increase the extent to which health services, individuals, and society are affected by MESBDs because of the forgone benefits of not investing in the next best alternative16–18,20–24,30. Corrective and rehabilitative health care services for MESBDs is critical in reducing the severity of lifelong disabilities and improving the quality of life for the affected children, as well as the economic productivity of the affected families1,2,9. Thus, cost analysis is of public health importance in influencing and informing health planning, policy decisions, resource allocations, informing further economic evaluations and assessing health system performance17,18,20,22,23,31,32. Consequently, the objective of this study was to conduct a cost analysis of the outpatient services for MESBDs from the providers’ viewpoints using an ingredient approach in selected hospitals in Kiambu County, Kenya.\n\n\nMethods\n\nThe study was conducted in four hospitals consisting of three county referral hospitals (Kiambu, Thika, and Gatundu), and PCEA Kikuyu orthopedic (faith-based) selected for providing corrective and rehabilitative outpatient health services to children born with MESBDs in the county. The three county referral hospitals were purposively selected being the only public hospitals providing these services in the county, whereas PCEA Kikuyu orthopedic hospital (faith-based) was selected by simple randomization using sealed envelopes between two faith-based hospitals for providing the same services in the county. A hospital-based cross-sectional study design was adopted to generate cost data from prevalence-based local morbidity data gathered using an ingredient approach to estimate the economic costs of corrective and rehabilitative outpatient health services from health care providers’ perspectives. This was however the best choice of study design for measuring the unit economic cost of health services as an attribute of the population, and thus provided a snapshot of the burden associated with the ‘silent’ public health problem and allowed for generalization of the study results in similar hospital settings in the region. Even though incidence data were readily available and easily accessible for the costing activity, prevalence-based data were excessively preferred to improve the accuracy of the profiled cost data and the estimation of the unit economic costs. This was an economic evaluation study, therefore was reported as per the CHEERS (checklist for consolidated health economic evaluation reporting standards) guidelines27.\n\nThe study population consisted of all children aged under five years old born to resident women of Kiambu County between January 1st, 2014, and December 31st, 2018, Cases were defined as live births with at least one clinically obvious major external structural birth defect referenced/or described by assistant occupational therapists and/or orthopedic surgeons and presented to the occupational therapy clinics for care from January 1st, 2018, to December 31st, 2018. Caregivers of children born with MESBDs were likely to seek outpatient corrective and rehabilitative health services at the study hospitals whether the children were born in or out of the county. Thus, the eligibility criterion defined above could minimize systemic bias and ensure the reliability of the study results.\n\nThe data for cost drivers were gathered retrospectively from health care providers’ perspective for a one-year time horizon between January 1st, 2018, and December 31st, 2018 for purposes of maintaining similar currency conversion, and inflation rates. Discounting for differential timing did not suffice in this study because the value of the resources considered for the analysis were categorized only as current costs.\n\nThe total (annual) economic costs were calculated for the defects (349 cases) for computing the unit economic costs as an average of the total costs expressed in Kenya Shillings (KES). The unit economic costs were calculated by dividing by the total annual costs by the number of cases using the following formula: -\n\n\n\nFurther, the unit economic costs were converted to United States Dollars ($) at an existing exchange rate of KES 98.00 equivalent to $1.00 in December 2018 using the following formula: -\n\n\n\nThis study assumed that the existing currency exchange rate of KES 98.00 in 2018 reflected the Purchasing Power Parity (PPP) globally. Thus, the unit economic costs for corrective and rehabilitative services for the defects were inflated to the U.S Dollar ($) Consumer Price Index (CPI) from January 2018 to December 201833.\n\nBefore data collection began, the Principal Investigator (PI) recruited and trained four nursing graduates as research assistants (RAs) to ensure that the data abstraction process that spanned from August 1st, 2019 to September 30th, 2019 was carried out in a standardized manner. We adopted an ingredient approach to retrospectively gather the prevalence-based data on caseloads for the cost analysis of corrective and rehabilitative outpatient health services. The cost ingredients of bracing, tendonectomy, and casting interventions quantified consisted of caseloads (morbidity data) by type of MESBDs, the number of braces, the number of bracings, the number of bracing review visits, the number of casting materials, the number of castings, the number of casting review visits, the number of tendonectomies, and the number of tendonectomies review visits considered as direct recurrent costs; and staff emoluments, staff-time, building space for rental, and utility charges categorized as overhead recurrent costs. The prevalence-based morbidity data were retrospectively drawn from outpatient occupational therapist registers; described as medical records containing information on health services provided to children with major external structural birth defects. The information captured in these registers includes dates of clinic visits, outpatient numbers, names of the patients, patients’ age, residence, diagnoses, and therapeutic interventions, among others. Following a predefined inclusion criterion, 349 cases were determined, and associated cost drivers identified, measured, and valued (quantified) using prevailing market prices and entered in a predetermined secondary data abstraction tool. On the other hand, staff-time for the hospitals’ executives comprising the medical superintendents, chief nursing officers, orthopedic surgeons, and health administrative officers were gathered through face-to-face enquires from the occupational therapists being the closest proxies for the officers mentioned above. The ingredient technique and prevalence-based data were chosen for possibly of generating detailed and improved accuracy of the profiled costing data. The data gathered comprised the following: -\n\nCaseloads/morbidity data: Following a predefined exclusion criterion, 349 cases of MESBDs were considered for the cost analyses.\n\nCasting: Castings used to stabilize the affected feet consisted of Plaster of Paris (POP) bandages, orthopedic cotton bandages, and glycerine valued at local market prices. A set of these materials valued at $3.9 were used to cast two cases of club foot. The study computed the number of castings and the number of revisits after the procedure for all cases of CTEV treated using these strategies.\n\nBraces: Braces consisted of leather foot covers, rubber soles, and metallic rods used to stabilize cases of club foot. Braces were sourced from the local markets as ready-made products, therefore, were valued at $15.31 using prevailing market prices. The study also enumerated the number of braces and the number of revisits after the procedure for all cases of CTEV treated using this strategy.\n\nThe number of tendonectomies: This was a procedure performed by surgeons to extend the Achilles tendon in club feet. This is largely an outpatient specialized procedure, therefore, existing market hospital charges for outpatient specialized surgical procedures were used to value the cost of tendonectomies estimated at $51.02. Similarly, the number of tendonectomies and the number of revisits after the procedure were computed for all the cases of CTEV that adopted this treatment strategy.\n\nEmolument for personnel: The personnel comprised assistant occupational therapists and support staff whose emolument were estimated based on the respective schemes for staff with at least ten years of work experience34–36. Emoluments for assistant occupational therapist consisted of basic salary, house allowance, commuter allowance, health risk allowance, and health extraneous allowance, whereas, for the support staff comprised basic salary, house allowance, and commuter allowance34–36. The monthly salary and benefits for an assistant occupational therapist at “Grade 10’’ was valued at $1,224.50, whereas support staff at “Grade 14’’ was valued at $173.50.\n\nRenting building space: Occupational therapy outpatient clinics were identified within the respective study hospitals whose plinth floor surface areas were measured in square feet and valued based on the existing local market rates for renting building spaces. The total renting space for the four hospitals was estimated at 3,593.63 square feet and valued at $0.37 local market value.\n\nUtility charges: Utilities included electricity; and water and sewerage estimated at $25.51 and $30.61 per month, respectively.\n\nSupervisory staff-time: The staff-time for the medical superintends/directors, orthopedic surgeons, chief nursing officers/directors, and health administrative officers/directors were identified and measured by consensus through face-face inquiries made to the assistant occupational therapists being the closest proxies for the above-mentioned officers. The staff-time for medical directors and orthopedic surgeons was measured as a single specialized medical practitioner’s consultation valued at $20.40 and quantified for five days a week for one calendar year. The staff-time for nursing directors and administrative directors on the other hand was measured as a single general medical practitioner’s consultation valued at $10.20 for five days a week for one calendar year.\n\nWe obtained ethical approval from Kenyatta National Hospital (KNH)-University of Nairobi (UoN) Ethics Review Committee (Ref. No: KNH-ERC/A/44). Data collected were de-identified using anonymous codes and entered in a laptop secured by an alphanumeric coded key known only to the PI to maintain confidentiality.\n\nPatient consent for publication: Patients were not directly involved because data was drawn from the medical registers, thus consent was not required.\n\nCase ascertainment, information, and systemic biases were expected in this study; therefore, the PI began by predefining an eligibility criterion (case definitions) for participation in the study and predetermining a secondary data abstraction tool for purposes of reducing case ascertainment biases. On the other hand, information biases were reduced by training the data collectors on secondary data extraction techniques from the outpatient occupational therapy clinics and entering data into the abstraction tools to ensure the process was conducted in a standardized manner. Further, all the registers for the entire one-year study period (2018) were reviewed and listed all the cases of external structural birth defects to reduce ascertainment and information biases in this study. Systemic bias was also reduced by excluding cases of delayed milestones, and/or developmental conditions due to management intervention similarities.\n\nFollowing data collection, filled secondary data abstraction tools were manually checked daily for accuracy and completeness and subsequently entered in a Microsoft Excel spreadsheet (Microsoft Office Professional Plus 2019) by two independent data managers to reduce potential errors. The PI cross-checked and validated the computerized dataset against predetermined data abstraction tools for analyses. Descriptive qualitative categorical variables were summarized in frequency tables, proportions, and percentages to show their distributions, whereas continuous variables were summarised and presented in means (averages). Costs assigned to the direct cost center consisted of; (i) the names and numbers of specific MESBDs (caseloads/morbidity data), (ii) the number of primary and review visits for castings and/or bracings and/or tendonectomies (iii) the number of assistant occupational therapists and their emoluments (iv) the number of support staff and their emoluments, and (v) the floor plinth area for renting. Overheads on the other hand comprised; (i) the number of orthopaedic surgeons and associated staff-time (ii) the number of medical superintendents/directors, and associated staff-time (iii) the number of chief nursing officers/directors and associated staff-time (iv) the number of health administrative officers/directors and associated staff-time, and (v) utility charges for electricity, water and sewerage. All the costs were categorized as recurrent and allocated to the direct cost center whereas overhead costs were shared proportionally among the respective types of MESBDs and allocated to the final costs center for estimation of the economic costs. The unit economic costs were calculated as average costs expressed in U.S Dollars and inflated to the U.S Dollar Consumer Price Index from January 2018 to December 2018 for all types of MESBDs collectively, and individual types of MESBDs. Capital costs did not suffice in this cost study because movable, and fixed capital resources were not considered for valuation because no inventory records existed for furniture, examination couches even though they appeared to have lost more than half of their economic half-lives, and capital donations in kind, whereas motor vehicles, motorcycles, and bicycles were not used either as direct, indirect, or intermediate costs for corrective and rehabilitative health services for the under-fives with MESBDs. The occupational therapy clinics on the other hand, also as fixed capital costs, were exceedingly small portions of the respective hospitals’ floor plinths, hence valued as recurrent costs using prevailing local market prices for building space rental.\n\nInflation factor and statistical uncertainties: All the resources were categorized as recurrent costs and inflated to the U.S Dollar ($) Consumer Price Index (Calculator) for a one-year time horizon from January 2018 to December 201833. This computation adjusted the unit economic costs to purchasing power parity as a factor of inflation, and minimized statistical uncertainties due to cost data scantiness and collection methods32,33. Consumer Price Index measures the mean changes in market prices over some time in which consumers pay for goods and services such as health services for MESBDs, thus was preferred because of being the best optimal measure of inflation in this study33.\n\n\nResults\n\nOf 349 cases 305 (87.39%) comprised congenital talipes equinovarus (CTEV) comprising unilateral congenital talipes equinovarus 300 (85.96%), bilateral congenital talipes equinovarus 3 (0.86%), unilateral congenital talipes equinovarus with germ valgus 1 (0.29%), and congenital talipes equinovarus with spina bifida 1 (0.29%). Additionally, the study observed 35 (10.03%) cases of congenital pes planus (CPP), and 9 (2.58%) cases of neural tube defects (NTD) consisting of hydrocephalus 5 (1.43%), spina bifida 3 (0.86%), and spina bifida with hydrocephalus 1 (0.29%) (Table 1).\n\nMESBDs, major external structural birth defects; %, percent\n\nResource quantification for casting materials costing $3.9 used for two procedures consisted of Plaster of Paris Bandage (7.6cm×2.7m×2pcs), orthopedic cotton bandage (15cm×3m×1pc), and glycerine oil (100 mililitres×1pc) costing $1.84, $1.22, and $0.82, respectively. A set of casting materials valued at $3.9 were used to cast two cases of club foot (Table 2).\n\ncm, centimetres; m, meters; mls, millilitres; pcs, pieces; $, USD\n\nThe annual economic costs were estimated and adjusted for inflation factors using the consumer price index calculator (CPI). Of the total unadjusted annual cost for all cases of the observed MESBDs ($392,436.49), almost two-thirds (71.48%) of resource inputs were accounted for by emoluments of occupational therapists, whereas administrative staff-time accounted for about one-quarter (18%) (Table 3).\n\n@ at; $, USD; %, percent\n\nOf the total unadjusted economic costs, overhead (shared) costs for these defects consisted of staff-time, staff emoluments, and utilities were estimated at $376,553.05 (Table 4). Of the total overhead costs ($376,553.05), emoluments for the occupational therapists accounted for three-thirds (74.11%), whereas staff-time costs accounted for 18.72% (Table 4).\n\n$, USD; %, percent\n\nOverheads were allocated proportionally (percentage-based) among the three specific cases: congenital talipes equinovarus (87.39%), congenital pes planus (10.03%), and spina bifida (2.58%) (Table 5).\n\n$, USD; %, percent\n\nOf the total unadjusted annual economic costs for major external structural birth defects; congenital talipes equinovarus was estimated at $343,959.87, whereas congenital pes planus and neural tube defects were estimated at $38,322.97 and $10,153.67, respectively (Table 6).\n\nCTEV, congenital talipes equinovarus; n, sub-total number of observations\n\nEstimation of adjusted economic costs: The unadjusted unit economic costs on the other hand were estimated at $1,124.46 for all the defects, $1,127.74 for congenital talipes equinovarus, $1,094.94 for congenital pes planus, and $ for neural tube defects $1,128.19 (Table 7). The annual and unit economic costs for all MESBDs observed were adjusted for the inflation factor to $397,765.72. The annual economic costs for congenital talipes equinovarus were estimated at $348,630.80, whereas congenital pes planus and neural tube defects were estimated at $38,843.39, and $10,291.56, respectively (Table 7). Adjusted unit economic costs on the other hand were estimated at $1,139.73 for all types of MESBDs, $1,143.05 for CTEV, $1,109.81 for CPP, and $1,143.51 for NTD (Table 7).\n\nMESBDs, major external structural birth defects; CTEV, congenital talipes equinovarus; CPP, pes planus; NTD, neural tube defects; CPI, consumer price index calculator\n\nThe study showed relatively similar unit economic costs of the defects despite wide variations among the caseloads for specific types of MESBDs (Figure 1).\n\n\nDiscussion\n\nTo our knowledge, this was the first study to estimate the unit economic costs of MESBDs from health care providers’ economic perspective among the under-five-year-old children in Kiambu County, Kenya. Substantial public health resources are continually allocated to the health care systems for care of children with MESBDs, however, the unit economic costs of care are barely known because they are rarely estimated mainly in the developing countries9,29. Sufficient access and utilization of corrective and rehabilitative health services remain an important public health intervention for improving the quality of life for birth defect-affected children globally1–4. Even though limited cost data, inadequate costing expertise, and the rarity of defects have been attributed to the lack of knowledge on their costs, it is of public health and economic interest to estimate the opportunity costs of health care services for MESBDs9,29. Worldwide, the results of this study could provide a baseline unit economic costs for the corrective and rehabilitative health services, inform efficient allocation of health resources, stimulate, and inform cost studies especially the costs’ arms of full economic evaluation analyses17,22,31.\n\nThe study encountered 349 cases consisting of 305 (87.39%) cases of CTEV, 35 (10.03%) cases of CPP, and nine (2.58%) cases of NTD. Congenital talipes equinovarus consisted of 300 (85.96%) cases of unilateral CTEV, three (0.86%) cases of bilateral CTEV, one (0.29%) case of unilateral CTEV with germ valgus, and one (0.29%) case of CTEV with spina bifida. Neural tube defects on the other hand comprised five (1.43%) cases of hydrocephalus, three (0.86%) cases of spina bifida, and one (0.29%) case of spina bifida with hydrocephalus. Despite variations in the number of cases (caseloads) observed for each of the defects mentioned above, this study showed a relatively similar unit economic cost for each defect in the county. The unit economic costs for NTD were approximated at $1,143.51, whereas CTEV, and CPP were valued at $1,143.05, and $1,109.81, respectively. Notably, the unit economic cost of providing corrective and rehabilitative outpatient health services for these defects collectively was approximately estimated at $1,139.73.\n\nDespite defects of the central nervous system contributing the least number (9) of cases compared to congenital talipes equinovarus (305), and congenital pes planus (35), its unit economic costs was relatively equivalent to the costs of the latter two types of the MESBDs observed in the county (Figure 1). Although some forms of neural tube defects are potentially fatal, the children who survive beyond infancy require substantial economic resources to deal with the related adverse health impacts1,2,9,15. The results of this study were indeed consistent with other research findings in the region and across the world that the greatest burden of disease associated with MESBDs is usually accounted for by the defects of the central nervous system11,15,37. The economic burden of spina bifida is usually substantial throughout the life of the affected individuals ascribed to the experienced high medical care expenditures in the early years of life with the defect and later reduced milestone development usually associated with spina bifida11,38. Our study similarly showed that neural tube defects followed by congenital pes planus accounted for the highest disease burden associated with MESBDs being shouldered by the health care systems in Kiambu county.\n\nEven though our study estimated the economic costs of these defects among the under-five-year-old children, our findings mimicked results of other studies such as in Germany where similarly high staggering economic costs were encountered among the general population with various forms of NTD between 2006 and 200911. Worldwide, spina bifida has singly been observed to account for the highest-burden of disease among other types of MESBDs11,15. Significant economic costs have been reported among new-born children with NTD during their first years of life, whereas, high healthcare expenditures have been observed during childhood, adolescents, and adulthood compared to the children without NTD globally11. In Germany, the average annual health expenditure of persons with spina bifida was estimated at €4,532, with inpatient health services contributing €1,358 (30.0%), outpatient health services €644 (14.2%), rehabilitation health services €29 (0.6%), drug therapy €562 (12.4%), and other remedies €1,939 (42.8%)11. In the United States among children aged between 1–17 years old, medical expenditures on spina bifida were estimated to cost 13 times as much as those on children without spina bifida37.\n\nNotably, annual direct economic costs of different forms of major birth defects were estimated at $2.6 billion in 2004 in the United States of America,5,39. Nonetheless, our study also endeavored to find and estimated the annual direct economic cost of MESBDs at $397,765.72. The defects encountered at the study hospitals consisted of neural tube defects, congenital talipes equinovarus, and congenital pes planus Despite different socioeconomic and demographic characteristics in Kenya and U.S, being developing, and developed countries respectively, this was indeed a remarkable empirical effort to estimate the direct economic costs of MESBDs in Kiambu County. Cost studies were pioneered in the United States of America by Dorothy Rice in 1967, and have since been undertaken widely in Europe and Australia unlike in middle-and low-income economies19. Low undertakings of cost studies, particularly in low-and middle-income economies have been attributed to the scarcity of data on the burden of these defects,20,28,29. Thus, the variations of annual direct economic costs could have been due to differences in the availability of the cost data, costing expertise, health services access, and utilization (economies of scale)5,17,19,39. Despite variations observed in the estimates of the economic costs, these findings point to the continuous disease burden associated with MESBDs in the county underpinning efficiency in resource utilization, and allocation for MESBDs in public and faith-based health facilities.\n\nThe few cases of NTD observed in this study could be attributed to a proportion of the carers of children with NTD seeking alternative therapies due to the associated adverse psychosocial effects experienced by the affected families38,40–42. Thus, the economic costs of NTD would be exponential compared to other forms of the defects observed in the study if all carers would have sought for care from the respective study hospitals. Nevertheless, the estimated costs demonstrated the potential catastrophic burden of the ‘silent’ economic problem in the region, thus underscores more scientific efforts to understand the magnitude of MESBDs regionally9. The observations made by this study have contradicted the epidemiological and economic fallacy that MESBDs are not of public health priority relative to other health events especially in resource-constrained countries1–4. Nevertheless, some limitations were inherent in this study; first and foremost, medical records used to draw the cost data were not designed for economic evaluation studies, whereas some of the defects were likely to delay childhood milestone development prolonging the demand for corrective and rehabilitative outpatient service possibly leading to more economic expenditures. The researchers also experienced difficulties in distinguishing the extent of the cost drivers for congenital talipes equinovarus occurring with spina bifida, congenital talipes equinovarus occurring with germ valgus, and spina bifida occurring with hydrocephalus, potentially due to inaccuracies of the profiled cost data.\n\n\nConclusions\n\nThis study estimated the economic costs of outpatient corrective and rehabilitative health services for MESBDs in Kiambu County in Kenya. Despite the fewest caseloads for the NTD, the study showed that NTD was associated with the highest burden of disease followed by CPP in the county. Despite CTEV proportionally contributing the highest caseload for the defects, it essentially accounted for the lowest burden of the disease associated with MESBDs in the county. This observation thus points to adverse developmental, and psychosocial impacts among the affected children and their families who are not able to access corrective and rehabilitative services. Similarly, these findings suggest a possible reduced economic productivity among the affected families arising from direct and indirect costs associated with major external structural birth defects. Therefore we would like to recommend further studies on the direct and indirect economic costs of MESBDs among children of school-going age to understand the impacts, and establishment of functional occupational therapy clinics in the ten sub-county hospitals to increase access of these services within Kiambu County.\n\n\nData availability\n\nHarvard Dataverse: Costing analysis of outpatient services for major external structural birth defects: An ingredient approach in selected hospitals in Kiambu County, Kenya. https://doi.org/10.7910/DVN/AULBDG43\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgment\n\nWe would like to acknowledge National Commission for Science, Technology, and Innovation (Ref. No. NACOSTI/P/19/75586/28325), Kiambu County Commissioner (Ref. No. ED. 12 (A)/1/VOL.11/107), and Kiambu County Health Department (Ref. No. KIAMBU/HRDU/AUTHO/2019/03/06/AgotGN) for permitting us to carry out this study in the county. We would also like to acknowledge the Medical Superintendents/Directors of the four hospitals for granting us the permission and support during data collection. Lastly, we would like to acknowledge the efforts of all data collectors in making this exercise a reality.\n\n\nReferences\n\nChristianson A, Howson CP, Modell B: March of Dimes: global report on birth defects, the hidden toll of dying and disabled children. March of Dimes: global report on birth defects, the hidden toll of dying and disabled children, 2005. Reference Source\n\nChristianson A, Howson CP, Modell B: March of dimes. Global report on birth defect The hidden toll of dying and disabled children. White Plains, New York, 2006. Reference Source\n\nWHO: Birth defects surveillance a manual for programme managers. World Health Organization, Centre for Disease Prevention and Control, and Internataional Clearinghouse for Birth Defects Surveillance and Research; 2014. Reference Source\n\nWHO: Birth defects surveillance: a manual for programme managers. World Health Organization, Centre for Disease Prevention and Control, and Internataional Clearinghouse for Birth Defects Surveillance and Research; 2020. Reference Source\n\nTinker SC, Gilboa S, Reefhuis J, et al.: Challenges in studying modifiable risk factors for birth defects. Curr Epidemiol Rep. 2015; 2(1): 23–30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFeldkamp ML, Carey JC, Byrne JLB, et al.: Etiology and clinical presentation of birth defects: population based study. BMJ. 2017; 357: j2249. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAndegiorgish AK, Andemariam M, Temesghen S, et al.: Neonatal mortality and associated factors in the specialized neonatal care unit Asmara, Eritrea. BMC Public Health. 2020; 20(1): 10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBhide P, Kar A: A national estimate of the birth prevalence of congenital anomalies in India: systematic review and meta-analysis. BMC Pediatr. 2018; 18(1): 175. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWaitzman NJ, Romano PS, Scheffler RM: Estimates of the economic costs of birth defects. Inquiry. 1994; 31(2): 188–205. PubMed Abstract\n\nWHO: Birth defects in South-East Asia a public health challenge situation analysis. World Health Organization, Regional office for South-East Asia: WHO Regional Office for South-East Asia; 2013. Reference Source\n\nBowles D, Wasiak R, Kissner M, et al.: Economic burden of neural tube defects in Germany. Public Health. 2014; 128(3): 274–81. PubMed Abstract \| Publisher Full Text\n\nBhide P, Gund P, Kar A: Prevalence of congenital anomalies in an Indian maternal cohort: healthcare, prevention, and surveillance implications. PLoS One. 2016; 11(11): e0166408. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFeldkamp ML, Reefhuis J, Mitchell AA, et al.: Understanding the causes of major birth defects: steps to prevention. 2015. Reference Source\n\nParker SE, Mai CT, Canfield MA, et al.: Updated national birth prevalence estimates for selected birth defects in the United States, 2004-2006. Birth Defects Res A Clin Mol Teratol. 2010; 88(12): 1008–16. PubMed Abstract \| Publisher Full Text\n\nWu VK, Poenaru D, Poley MJ: Burden of surgical congenital anomalies in Kenya: a population-based study. J Trop Pediatr. 2013; 59(3): 195–202. PubMed Abstract \| Publisher Full Text\n\nSimeone RM, Feldkamp ML, Reefhuis J, et al.: CDC Grand Rounds: understanding the causes of major birth defects - steps to prevention. MMWR Morb Mortal Wkly Rep. 2015; 64(39): 1104–7. PubMed Abstract \| Publisher Full Text\n\nDrummond MF, Sculpher MJ, Claxton K, et al.: Methods for the economic evaluation of health care programmes. Oxford university press; 2015. Reference Source\n\nKirigia JM: Economic evaluation of public health problems in sub-Saharan Africa. University of Nairobi; 2009. Reference Source\n\nMugisha F, Kouyate B, Dong H, et al.: Costing health care interventions at primary health facilities in Nouna, Burkina Faso. Afr J Health Sci. 2002; 9(1–2): 69–79. PubMed Abstract \| Publisher Full Text\n\nConteh L, Walker D: Cost and unit cost calculations using step-down accounting. Health Policy Plan. 2004; 19(2): 127–35. PubMed Abstract \| Publisher Full Text\n\nMcIntosh E: Using discrete choice experiments within a cost-benefit analysis framework: some considerations. Pharmacoeconomics. 2006; 24(9): 855–68. PubMed Abstract \| Publisher Full Text\n\nCunningham SJ: Economic evaluation of healthcare--is it important to us? Br Dent J. 2000; 188(5): 250–4. PubMed Abstract \| Publisher Full Text\n\nSandmann FG, Robotham JV, Deeny SR, et al.: Estimating the opportunity costs of bed‐days. Health Econ. 2018; 27(3): 592–605. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWalker D, Kumaranayake L: Allowing for differential timing in cost analyses: discounting and annualization. Health Policy Plan. 2002; 17(1): 112–8. PubMed Abstract \| Publisher Full Text\n\nMogyorosy Z, Smith P: The main methodological issues in costing health care services: a literature review. Centre for Health Economics, University of York Working Papers. 2005. Reference Source\n\nMogyorosy Z, Smith P: The main methodological issues in costing health care services: a literature review. 2005. Reference Source\n\nHusereau D, Drummond M, Petrou S, et al.: Consolidated Health Economic Evaluation Reporting Standards (CHEERS)--explanation and elaboration: a report of the ISPOR Health Economic Evaluation Publication Guidelines Good Reporting Practices Task Force. Value Health. 2013; 16(2): 231–50. PubMed Abstract \| Publisher Full Text\n\nGedefaw A, Teklu S, Tadesse BT: Magnitude of neural tube defects and associated risk factors at three teaching hospitals in Addis Ababa, Ethiopia. Biomed Res Int. 2018; 2018: 4829023. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKhurmi MS, Gupta M, Chaudhari G: Addressing the public health challenge of birth defects in India. Indian J Child Health. 2014; 1(3): 95–8. Publisher Full Text\n\nPreedy VR, Watson RR: Handbook of disease burdens and quality of life measures. Springer; 2010. Publisher Full Text\n\nBirch S, Gafni A: Cost-Effectiveness and Cost Utility Analyses: Methods for the Non-Economic Evaluation of Health Care Programmes and How We Can Do Better. Managing Technology in Healthcare: Springer; 1996: 51–67. Publisher Full Text\n\nBriggs A, Sculpher M, Buxton M: Uncertainty in the economic evaluation of health care technologies: the role of sensitivity analysis. Health Econ. 1994; 3(2): 95–104. PubMed Abstract \| Publisher Full Text\n\nbls.gov/inflation_calculator.htm. Retrieved on 13/10/2020. 2020.\n\nGoK: Revised scheme of service for assistant occupational therapists and occupational therapists. Nairobi, Kenya: Government Printer. 2014. Reference Source\n\nSRC: Salaries and remuneration commission circular on allowances in the public service. Nairobi, Kenya: Government Printer. 2014.\n\nSRC: Salaries and remuneration circular on health workers allowances. Nairobi, Kenya: Government Printer. 2015.\n\nOuyang L, Grosse SD, Armour BS, et al.: Health care expenditures of children and adults with spina bifida in a privately insured U.S. population. Birth Defects Res A Clin Mol Teratol. 2007; 79(7): 552–8. PubMed Abstract \| Publisher Full Text\n\nRofail D, Maguire L, Kissner M, et al.: A review of the social, psychological, and economic burdens experienced by people with spina bifida and their caregivers. Neurol Ther. 2013; 2(1–2): 1–12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMburia-Mwalili A, Yang W: Birth defects surveillance in the United States: Challenges and implications of international classification of diseases, tenth revision, clinical modification implementation. Int Sch Res Notices. 2014; 2014: 212874. PubMed Abstract \| Publisher Full Text\n\nKronenberger WG, Thompson Jr RJ: Psychological adaptation of mothers of children with spina bifida: Association with dimensions of social relationships. J Pediatr Psychol. 1992; 17(1): 1–14. PubMed Abstract \| Publisher Full Text\n\nWallander JL, Feldman WS, Varni JW: Physical status and psychosocial adjustment in children with spina bifida. J Pediatr Psychol. 1989; 14(1): 89–102. PubMed Abstract \| Publisher Full Text\n\nVermaes IPR, Janssens JMAM, Bosman AMT, et al.: Parents' psychological adjustment in families of children with spina bifida: a meta-analysis. BMC Pediatr. 2005; 5: 32. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAgot GN: Costing analysis of outpatient services for major external structural birth defects: An ingredient approach in selected hospitals in Kiambu County, Kenya. Harvard Dataverse. 2021. http://www.doi.org/10.7910/DVN/AULBDG" }	[ { "id": "88083", "date": "16 Aug 2021", "name": "Vincent Okungu", "expertise": [ "Reviewer Expertise Health economics & policy" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study design is confusing:\nThe design is not cross-section but rather retrospective ingredient-based approach using secondary data from purposively selected hospitals.\n\nAuthors need to itemize cost drivers; identify major cost drivers for each intervention.\n\nWhat are cost centers?\n\nThe study is purely concerned with operational costs/recurrent. I fail to see any element of economic costing.\n\nAuthors need to clearly define economic costing, and how it fits with this type of study.\n\nWhy consider inflation factor when the time horizon is just one year? Doesn't make sense to me.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "7042", "date": "20 Aug 2021", "name": "George Agot", "role": "Author Response", "response": "1. We adopted an ingredient-based approach as a technique for gathering resources inputs for economic evaluation studies, both partial and full, however, cross-sectional design was specified to demonstrate the dimension of data collection. i.e., data was collected at a point in time similar to the retrospective dimension of data collection. 2. The interventions are clearly specified in this study as castings, bracings, and tendonectomies whose cost drivers are specifically stated as the number of the interventions, the number of first visits, the number of revisits among others including quantification of the resource inputs for casting, bracings and tendonectomy procedures. Staff emolument was identified as the major cost driver for these interventions thus will be included in the results section of the revised version of this article. 3. The cost centers are indeed not specified, however, they will be included in the revised version of this article to illustrate the resources assigned to the direct, intermediate, and indirect cost centers and their allocations to the final cost center. 4. This study is characteristically a cost analysis study in my opinion demonstrated by the methods used to determine the monetary value of the resource inputs, for example, the prevailing market prices and opportunity costs (staff-time) where applicable. Operational costs are usually established by financial cost analysis techniques to determine the actual expenditures on specific resource inputs for a service/good; thus not to determine the opportunity (economic) costs. Nevertheless, both financial and economic costs can be described as recurrent financial costs and recurrent economic costs respectively. 5. As explained in number four above, this study is purely an economic cost analysis based on the attributes stated above. However, I would like to point out that economic cost analysis and economic costing analysis have different meanings to some extent in my view. Economic cost analysis determines the opportunity costs (the best-forgone alternative choice) of the expenditures already incurred whereas, economic costing analysis endeavors largely to determine the opportunity cost of expenditures likely to be incurred. 6. Discounting for differential timing is indeed not required in this study owing to the one-year time horizon as specified in the introduction section of this article. This explanation will be corrected in the methods and results section in the revised version of this article. However, computer price index (CPI) calculation was adopted particularly in this study as a strategy for uncertainty analysis attributed to the scantiness of the cost data and data collection methods." }, { "c_id": "7053", "date": "20 Aug 2021", "name": "George Agot", "role": "Author Response", "response": "To further clarify point number four, operational costs are essentially determined by accounting procedures as actual expenditures." } ] } ]	1	https://f1000research.com/articles/10-359
https://f1000research.com/articles/10-58/v1	01 Feb 21	{ "type": "Case Report", "title": "Case Report: Diagnostic challenge of COVID-19 associated pulmonary aspergillosis (CAPA)", "authors": [ "Hanan Albasata", "Maha M. Alamri", "Saud A. Almuhaidb", "Abdullah M. Aljebreen", "Reem S. Almaghrabia", "Maha M. Alamri", "Saud A. Almuhaidb", "Abdullah M. Aljebreen", "Reem S. Almaghrabia" ], "abstract": "Background: Coronavirus disease 2019 (COVID-19) was declared a pandemic in March 2020 by the World Health Organization (WHO). Severe COVID-19 is represented with acute respiratory distress syndrome (ARDS) that requires mechanical ventilation. Moreover, recent studies are reporting invasive fungal infection associated with severe COVID-19. It is unclear whether the prescription of immunotherapies such as corticosteroids, or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection itself is risk factor for COVID-19-associated invasive pulmonary aspergillosis (CAPA). Hence, fungal infections present an additional uncertainty in managing COVID-19 patients and further compromise the outcome. Case study: Here we report a case of SARS-CoV-2 complicated by invasive pulmonary aspergillosis (IPA) in a patient with no traditional risk factors for IPA. Admitted to ICU due to ARDS on mechanical ventilation, the patient deteriorated clinically with unexplained increased of fraction of inspired oxygen (FiO2) requirement from 50% to 80%. Investigations showed borderline serum galactomannan, nonspecific radiological findings reported to be atypical for COVID-19, and the respiratory sample grew Aspergillus spp. Main diagnosis: COVID-19 related fungal infection. The patient was treated with antifungal therapy for four weeks. He improved clinically after one week of starting antimicrobial treatment. After a prolonged ICU stay (87 days) due to infection control precaution, he was discharged from the ICU and moved to a long-term facility for further management and support. Conclusions: This case highlights the diagnostic challenge in such cases. and the importance of early recognition of CAPA which can optimize therapy by administration of appropriate antifungal agents that may impact mortality.", "keywords": [ "COVID", "SARS-CoV2", "Aspergillosis", "Invasive", "Pulmonary", "Critical." ], "content": "Background\n\nInvasive pulmonary aspergillosis (IPA) is typically thought to cause disease in immunocompromised hosts, particularly in neutropenic patients. In the last two decades, it has been more commonly recognized in critically ill patients, particularly those with severe acute respiratory distress syndrome (ARDS)1. An increasing number of IPA cases complicating severe influenza have been reported following the H1N1 influenza pandemic in 2009. Vanderbeke et al. described 128 cases published between 1952–20182. In late December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged as a public health emergency and has spread globally3. Co-infections among patients with coronavirus disease 2019 (COVID-19) are currently well described in the literature. Chlamydophila pneumoniae, Mycoplasma pneumoniae, and human metapneumovirus are among the common pathogens identified4. Intensive care unit admission is required in around 5–30% of patients with COVID-19. IPA has started to be recognized in severe COVID-19 infection, with multiple case series of severe COVID-19 pneumonia complicated by IPA having been reported since the start of the pandemic5–10. Here we report a case of IPA that was diagnosed in a patient with severe COVID-19 infection.\n\n\nCase\n\nWe report the case of a 29-year-old Saudi male security guard known to have diabetes mellitus and chronic kidney disease. He was admitted to hospital at the end of May 2020 as a case of COVID-19 pneumonia that was complicated with diabetic ketoacidosis and required admission to the intensive care unit (ICU). He rapidly deteriorated with progression to ARDS, requiring intubation and mechanical ventilation. He also suffered a cardiac arrest that required resuscitation for 12 minutes. He received Lopinavir/Ritonavir and Ribavirin along with Ceftriaxone in the referring hospital (dosage unknown). He was then transferred to the King Faisal Specialist Hospital and Research Centre (KFSHRC) around six days after his initial diagnosis for further management.\n\nOn day 0 of ICU admission at KFSHRC the patient was deeply sedated on intravenous (IV) propofol 150mg/hour and fentanyl 100mcg/hour, measured temperature 34.7 °C, intubated on pressure control ventilation requiring FiO2 50% and positive end-expiratory pressure (PEEP) 8 cm H2O. He required IV norepinephrine 0.02– 0.2 mcg/kg/min to maintain his mean arterial blood pressure above 65 mmHg. His blood workup on day 0 was white blood cell count (WBC) 8.28 ×109 / L (3.9–11 × 109 / L), absolute lymphocytes count 0.5 ×109 / L (1.50– 4.30 × 109 / L), platelet 54 ×109 / L (155– 435 × 109 / L), creatinine 603 umol/L (64 – 115 umol/L), and galactomannan antigen (AG) 0.48 (>0.5 reactive). A beta D-glucan test was not done as it was not available.\n\nThe patient was started on continuous renal replacement therapy (CRRT); his clinical status deteriorated with increased ventilation requirement of FiO2 50% to 80%, PEEP 8 to 16 cm H2O on day 4. A chest X-ray showed multiple bilateral ill-defined patchy opacities in the right lower lung zone (Figure 1). A computed tomography (CT) scan for his chest was done and showed multiple bilateral patchy ground-glass opacities (Figure 2, Figure 3). Bilateral lower lobe consolidations with air bronchogram showed greater involvement of the right lower lobe, while an unenhanced CT of the brain demonstrated hyperdense foci seen in the left inferior frontal, right parietal lobes with surrounding edema, and right central sulcus compatible with intra-parenchymal hemorrhage and subarachnoid hemorrhage, respectively. A follow-up MRI of the brain was obtained, showing an increased gyral pattern of T1 and FLAIR sequence, as observed in the bilateral occipital, bilateral frontal, and right parietal lobes, likely related to laminar necrosis from the anoxic- ischemic event. Gradient recalled echo (GRE) sequence showed scattered areas of blooming artifacts that are likely to be related to recent extensive hemorrhage. The brain findings were suggestive of hemorrhage and hypoxic injuries of vascular causes of previous cardiac arrest events.\n\nThe radiograph shows multiple bilateral ill-defined patchy opacities more in the right lower lung zone.\n\nThe scan shows a right middle lobe central ground-glass density with surrounding consolidation (arrow), giving the appearance of reversed halo sign 'atoll-sign'. A peripheral ground-glass density (curved arrow) is seen in the right lower lobe; a typical picture of coronavirus disease 2019 (COVID-19).\n\nThe scan shows a left lower lobe nodule (arrow) with surrounding faint ground-glass densities giving halo sign appearance.\n\nOn day 0, the patient was started on hydroxychloroquine 400mg orally every 12 hours for 1 day, followed by a maintenance dose of 200mg every 12 hours and azithromycin 500mg orally once followed by a maintenance dose of 250mg daily, respectively, for a total duration for 5 days. His antimicrobial therapy was escalated to Meropenem 0.5 gm IV every 12 hours. He continued to worsen, and thus his septic screen was repeated. His blood and urine culture remained negative.\n\nTracheal aspirate culture on day 0 grew Aspergillus fumigatus and Aspergillus flavus (Figure 4a, Figure 4b, Figure 5a, Figure 5b). Bronchoscopy was considered; however, it was not done due to concerns of COVID-19 transmission to the house staff. The patient was then started on dual antifungal therapy (day 4) for 1 week; Voriconazole 400mg orally every 12 hours as a loading dose, then 200mg every 12 hours as maintenance. In addition, the patient was given Caspofungin 70mg IV as a loading dose, followed by 50mg daily. After this Voriconazole monotherapy therapy (200mg orally every 12 hours) for a total duration of four weeks was completed in the hospital. One week after starting antifungal therapy (day 10 in the ICU), the secretions improved and the ventilator setting was decreased to FiO2 30%. Unfortunately, the patient on day 5 of ICU admission was found to be in a vegetative state secondary to anoxic brain damage post-cardiac arrest. The Glasgow Coma Scale was GCS 10/15 on tracheostomy.\n\nOlive-lime green colony morphology on sabouraud’s dextrose agar.\n\nRadiate, biseriate conidia.\n\nGray-green colony morphology on sabouraud’s dextrose agar.\n\nUniseriate conidiophore with columnar conidia.\n\nThe patient had a prolonged ICU stay due to infection control precaution; his stay was complicated with rhabdomyolysis, difficulty to wean him from ventilation, nosocomial infection after 30 days of ICU admission, and persistent COVID-19 virus shedding up to 73 days. The patient was then moved to a long-term facility (on day 87) after discharging him from the ICU.\n\n\nDiscussion\n\nThis case further supports the association between IPA and severe COVID-19 infection. It highlights the importance of early diagnosis and treatment of this serious complication that can impose increased mortality. The diagnosis of IPA in patients in ICU without classical risk factors like neutropenia remains challenging1–9.\n\nMultiple studies from China have reported different rates of Aspergillus infections among patients with COVID-19. The estimated rates of Aspergillus co-infection in these combined studies are as follows: in Jiangsu province, 60/257 (23.3%), Zhejiang province, 8/104 (7.7%); and lastly, Wuhan, 13/48 (27%). All these reported CAPA cases lack standardization in diagnostic criteria and use specific definitions to identify and define CAPA10,11. A European case series reported severe COVID-19 pneumonia complicated by IPA. All 27 cases were for patients admitted to the ICU, the majority of whom were intubated. The median duration between CAPA diagnosis and symptom onset was six days. Aspergillosis diagnosis was as early as three days post ICU admission or as late as 28 days10. Of the 27 patients, 12 (44%) received corticosteroids during their ICU stay, and 19 (70%) were treated with mould-active antifungal medication5–8. Bartoletti and colleagues found 22 (73%) of the 30 patients who were diagnosed with CAPA received Tocilizumab, and 18 (60%) received corticosteroids9.\n\nMixed fungal infection similar to our case was reported in two patients in a study from Pakistan, one with A. falvus and A. famigatus diagnosed as CAPA, while a second patient was thought to be colonizned with A. falvus and A. nigar12. Diagnosis is challenging due to the difficulty in differentiating between colonization and active disease in positive culture cases. Recently Arastehfar et al. suggested that galactomannan (GM) testing of bronchoalveolar fluid (BALF), or even of tracheal aspirates, may support CAPA's diagnosis11, though the test has not been validated for these specimens and cut-off values are not yet established.\n\nSpanish tertiary hospitals carried out a retrospective study in patients with confirmed SARS-CoV-2 by PCR who isolated Aspergillus spp. in respiratory samples from bronchial aspirates (BAS) and bronchoalveolar lavages (BAL). Galactomannan assays were performed in serum and/or BAL with a cut-off index of 0.5 for both samples. COVID-19 associated IPA cases were classified according to the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group EORTC/MSG criteria13 and Aspergillus algorithm for use in critically ill patients AspICU algorithm14. Aspergillus spp. were grown in ten patients from respiratory samples. Seven patients were intubated in the ICU. All isolates were detected from deep respiratory samples: eight BAS one sputum and one BAL. Galactomannan analyses were run in only three patients, one with positive values in both serum and BAL ((1.97, 3.87) and another with repeated positive values in BAL samples (2.16, 1.11). The third patient had a negative serum galactomannan test (0.22)15.\n\nPerforming bronchoscopy and obtaining a bronchial wash is challenging in patients with COVID-19. It carries a significant risk of transmission to healthcare workers, which further complicates the diagnosis of IPA in this patient population16. A surveillance strategy for fungal co-infections in intubated patients with COVID-19 was done by Brown et al.17 utilizing weekly serum1–3-β-D-glucan, galactomannan Aspergillus enzyme immunoassay (EIA) and Aspergillus PCR from bronchoalveolar lavage or endotracheal aspirates. A total of 62 patients were examined, and a galactomannan test of tracheal aspirates was performed for 85 samples; positive results were seen in six out of 62 patients, of which positive Aspergillus PCR was seen in five out of the six, and two grew Aspergillus fumigatus in culture. CAPA was clinically suspected in two patients. One of these patients’ GM from BAL was not performed. Whether galactomannan positivity of endotracheal aspirates is a marker for CAPA or reflects upper airway colonization is not clear. It should be noted that none of their cases met the definition of CAPA17.\n\nThe radiological differentiation between IPA and COVID-19 is often complex, as the radiological changes in IPA in non-neutropenic patients are diverse and non-specific. For instance, ground-glass opacities and dense consolidation are often found in COVID-19 and IPA18–20.\n\nTo the best of our knowledge, this is the first reported case of mixed fungal infection in COVID-19 in the Middle East region. This case report further supports published data about severe COVID-19 and invasive fungal infection. Diagnosis of IPA in critically ill patients with COVID-19 in ICUs remains a challenge. The difficulties we faced were that the CT chest scan showed left lower lobe nodules with surrounding faint ground-glass densities which are not sufficient to define CAPA, while tracheal aspirate cultures positive for the presence of A. fumigatus and A. flavus cannot differentiate between colonization or true invasion. Obtaining future tests from BAL to support the diagnosis is complicated by the restriction in preforming bronchoscopy in patients with COVID-19 due to the risk of aerosol generation. The accuracy of serum galactomannan to diagnose IPA would increase if consecutive tests were performed to override its poor sensitivity in IPA detection in non-neutropenic patients in ICUs. Lastly, repeated chest imaging to assess clinical response of the patient with IPA after completion of antifungal therapy unfortunately was not done, as the patient was kept on conservative and minimal intervention management.\n\n\nConclusions\n\nIPA can complicate severe COVID-19 pneumonia. The diagnosis of CAPA is often challenging and requires a high index of suspicion. A constellation of clinical, biochemical, microbiological, and radiological criteria needs to be incorporated to establish the diagnosis. Timely diagnosis and management are required for better outcomes.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the relative of the patient.", "appendix": "References\n\nBlot S, Rello J, Koulenti D: Diagnosing invasive pulmonary aspergillosis in ICU patients: putting the puzzle together. Curr Opin Crit Care. 2019; 25(5): 430–437. PubMed Abstract \| Publisher Full Text\n\nVanderbeke L, Spriet I, Breynaert C, et al.: Invasive pulmonary aspergillosis complicating severe influenza: epidemiology, diagnosis and treatment. Curr Opin Infect Dis. 2018; 31(6): 471–480. PubMed Abstract \| Publisher Full Text\n\nHuang C, Wang Y, Li X, et al.: Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet. 2020; 395(10223): 497–506. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLai CC, Wang CY, Hsueh PR: Co-infections among patients with COVID-19: The need for combination therapy with non-anti-SARS-CoV-2 agents? J Microbiol Immunol Infect. 2020; 53(4): 505–512. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRutsaert L, Steinfort N, Van Hunsel T, et al.: COVID-19-associated invasive pulmonary aspergillosis. Ann Intensive Care. 2020; 10(1): 71. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlanio A, Dellière S, Fodil S, et al.: Prevalence of putative invasive pulmonary aspergillosis in critically ill patients with COVID-19. Lancet Respir Med. 2020; 8(6): e48–e49. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKoehler P, Cornely OA, Böttiger BW, et al.: COVID-19 associated pulmonary aspergillosis. Mycoses. 2020; 63(6): 528–534. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvan Arkel ALE, Rijpstra TA, Belderbos HNA, et al.: COVID-19-associated Pulmonary Aspergillosis. Am J Respir Crit Care Med. 2020; 202(1): 132–135. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBartoletti M, Pascale R, Cricca M, et al.: Epidemiology of invasive pulmonary aspergillosis among COVID-19 intubated patients: a prospective study. Clin Infect Dis. 2020; ciaa1065. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPemán J, Ruiz-Gaitán A, García-Vidal C, et al.: Fungal co-infection in COVID-19 patients: Should we be concerned? Rev Iberoam Micol. 2020; 37(2): 41–46. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nArastehfar A, Carvalho A, van de Veerdonk FL, et al.: COVID-19 Associated Pulmonary Aspergillosis (CAPA)-From Immunology to Treatment. J Fungi (Basel). 2020; 6(2): 91. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNasir N, Farooqi J, Mahmood SF, et al.: COVID-19-associated pulmonary aspergillosis (CAPA) in patients admitted with severe COVID-19 pneumonia: An observational study from Pakistan. Mycoses. 2020; 63(8): 766–770. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDonnelly JP, Chen SC, Kauffman CA, et al.: Revision and Update of the Consensus Definitions of Invasive Fungal Disease From the European Organization for Research and Treatment of Cancer and the Mycoses Study Group Education and Research Consortium. Clin Infect Dis. 2020; 71(6): 1367–1376. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlot SI, Taccone FS, Van den Abeele AM, et al.: A Clinical Algorithm to Diagnose Invasive Pulmonary Aspergillosis in Critically Ill Patients. Am J Respir Crit Care Med. 2012; 186(1): 56–64. PubMed Abstract \| Publisher Full Text\n\nFalces‐Romero I, Ruiz‐Bastián M, Díaz‐Pollán B, et al.: Isolation of Aspergillus spp. in respiratory samples of patients with COVID-19 in a Spanish Tertiary Care Hospital. Mycoses. 2020; 63: 1144–1148. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWahidi MM, Lamb C, Murgu S, et al.: American Association for Bronchology and Interventional Pulmonology (AABIP) Statement on the Use of Bronchoscopy and Respiratory Specimen Collection in Patients With Suspected or Confirmed COVID-19 Infection. J Bronchology Interv Pulmonol. 2020; 27(4): e52–e54. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrown LAK, Ellis J, Gorton R, et al.: Surveillance for COVID-19-associated pulmonary aspergillosis. Lancet Microbe. 2020; 1(4): e152. Publisher Full Text\n\nShi H, Han X, Jiang N, et al.: Radiological findings from 81 patients with COVID-19 pneumonia in Wuhan, China: a descriptive study. Lancet Infect Dis. 2020; 20(4): 425–434. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShaghaghi S, Daskareh M, Irannejad M, et al.: Target-shaped combined halo and reversed-halo sign, an atypical chest CT finding in COVID-19. Clin Imaging. 2021; 69: 72–74. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGeorgiadou SP, Sipsas NV, Marom EM, et al.: The Diagnostic Value of Halo and Reversed Halo Signs for Invasive Mold Infections in Compromised Hosts. Clin Infect Dis. 2011; 59(9): 1144–1155. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "79238", "date": "24 Feb 2021", "name": "Ali S. Omrani", "expertise": [ "Reviewer Expertise Infectious diseases in immune compromised hosts and critically ill patients" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAn interesting report on an emerging clinical problem in the context of the ongoing SARS-CoV-2 pandemic. The authors describe the case in reasonable detail, its clinical progression and outcome. The discussion included a good contextualization of the case in the light of the salient knowns in the area. I suggest the authors consider the following comments to further improve their manuscript:\nThe first letter of an antimicrobial agent's genetic name should not be capitalized when mentioned mid-sentence (examples from the manuscript: caspofungin, voriconazole, meropenem, lopinavir/ritonavir, ribavirin, and ceftriaxone).\n\nSARS-Co-2 is the virus, while COVID-19 is the disease. The following expressions are incorrect: \"COVID-19 transmission\", \"persistent COVID-19 virus shedding\", \"COVID-19 infection\".\n\nWas the patient intubated in the referring hospital? If so, what was the timeframe between intubation and day 0 ICU in KFSHRC?\n\nDid the patient receive corticosteroids, tocilizumab, sarilumab, or baricitinib in the referring hospital or in KFSHRC? It should be possible to obtain those details from the referring hospital? State if none was given.\n\nIt would be useful to comment in the discussion on the isolation of Aspergillus species from baseline BAL (day 0).\n\nIt would be useful to comment on combination AFT for IPA.\n\nWhat are the prevailing Aspergillus species in Saudi Arabia? Is the isolation of A. flavus unusual?\n\nAre there any other reports of CAPA from the Middle East or the Gulf region?\n\nThe article below is a comprehensive review of CAPA reports up to August 20201. The authors might find it useful for their discussion.\nThank you\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [ { "c_id": "6450", "date": "07 May 2021", "name": "Hanan AlBasata", "role": "Author Response", "response": "Thank you for your review and comments, the following has been done in response to your valuable input: Comment 1: edited. Comment 2: Changed. Comment 3: added. Comment 4: Yes, he did receive dexamethasone in KFSH, added to the manuscript. Comment 5: Added. Comment 6: Added. Comment 7: A paragraph was added for the most prevalent spp. locally and globally. Comment 8: At the time of the writing no reported cases, however recently a case series in Kuwait was published and this was added in the discussion." } ] } ]	1	https://f1000research.com/articles/10-58
https://f1000research.com/articles/7-1951/v1	19 Dec 18	{ "type": "Research Article", "title": "Comparison of tillage costs among eight paddy farm regions in East Kalimantan, Indonesia", "authors": [ "Karmini Karmini" ], "abstract": "Background: Tillage is done to prepare land for wetland paddy farming, and it is commonly done by hand tractor. The purposes of this study were to identify the levels of ownership of hand tractor by paddy farmers, to describe the rental of hand tractor in rural areas, to calculate and compare the tillage costs on eight paddy farm regions, and to understand the utilization of farm machinery for paddy farming in East Kalimantan, Indonesia. Methods: The study areas were Subcities/Subdistricts of North Bontang, South Bontang, Muara Muntai, Loa Janan, Tenggarong Seberang, Waru, Penajam, and Babulu. Data collection was done by interviewing 380 respondents. Analysis of data used the Chi Square test. Results: The number of hand tractor renters (87.37%) in East Kalimantan 2014 was bigger than that of hand tractor owners (12.63%). The tillage costs in Tenggarong Seberang, Loa Janan, and Muara Muntai in 2014 were IDR700,000.00 ha-1, IDR750,000.00 ha-1, and IDR700,000.00 ha-1, respectively. Tillage costs were the same in Babulu, Penajam, Waru, South Bontang, and North Bontang (IDR1,000,000.00 ha-1 in each district). Conclusions: There are significant differences the number of hand tractor owners, the number of hand tractor renters, and the tillage costs among the eight paddy farm regions in East Kalimantan, Indonesia.", "keywords": [ "Cost", "East Kalimantan", "land", "paddy farm", "tillage." ], "content": "Introduction\n\nWetland paddy farming in East Kalimantan is a method of modern farming in which paddy farmers commonly by using hand tractor in land preparation. Tillage cost has important role in cost structure of paddy farming. The tillage cost can vary significantly, and negatively affects paddy farm income in East Kalimantan, Indonesia (Karmini, 2017). The increase of tillage cost leads the increase of production cost of paddy farming and the decrease of paddy farm income and household income of paddy farmers. This is supported by the research result of Larson & Plessmann (2009).\n\nWetland paddy farming is done in most regions in East Kalimantan. Information is needed about the tillage cost in different paddy farm areas to formulate policy on farm machinery utilization in specific areas containing paddy farms. The purposes of this study were to identify the ownership of hand tractor by paddy farmers, to describe the rental of hand tractors in rural areas, to calculate and compare the tillage costs on eight paddy farm regions, and to know the machinery utilization for paddy farming in East Kalimantan, Indonesia. The hypotheses of this study were that there are no significant differences the number of hand tractor owners, the number of hand tractor renters, and the tillage costs among the eight paddy farm regions in East Kalimantan, Indonesia.\n\n\nMethods\n\nThis study was held from November 2013 to April 2014 in Province of East Kalimantan, Republic of Indonesia. The determination of study areas based on two-stage clustered sampling. The study areas were Bontang City (North Bontang and South Bontang), Kutai Kartanegara District (Muara Muntai, Loa Janan, and Tenggarong Seberang), and Penajam Paser Utara District (Waru, Penajam, and Babulu).\n\nThe overall population in the areas examined in this study was 36,970 households of paddy farmers. The minimum sample size for populations of 20,000 and 50,000 people is 377 and 382, respectively (Rea & Parker, 1997). The sample size used in this study was therefore 380 respondents. The determination of the number of respondents in each study areas was based on proportional sampling.\n\nPurposive sampling was applied in selecting respondents. Inclusion criterias for respondents were farmers who are currently engaged in wetland paddy farming, lived minimum 5 years in study area, and had experience continuously minimum 2 years cultivate paddy. Exclusion criterias for respondents were lived less than 5 years in study area and had experience less than 2 years cultivate paddy but farmers have own land or become labors. Respondents were paddy farmers who are currently engaged in wetland paddy farming. The researcher went to paddy field and met with potential respondents in person, then provided information on the purposes of study and the right of them to not answer the questions at any time and assured that the data would be kept confidential and only aggregate data would be used. After they gave the consent to be interviewed, they were given the choice to decide the place for the interviews using the questionnaire (Karmini, 2018), either at home or other places which were convenient for them. Both the researcher and respondents discussed directly at the same place. This study was approved by Head of Department of Agribusiness, Faculty of Agriculture, University of Mulawarman (Tetty Wijayanti, SP, MP; approval number 2104/UN17.3/TU/2013). Each participant gave their written informed consent to participate in the study.\n\nThis study performed analysis by using the software of IBM SPSS Statistics 20 and tested hypotheses using the Chi Square test with α = 0.05.\n\n\nResults and discussion\n\nAll 380 respondents completed the questionnaire in full. From our study, we observed that paddy farmers in East Kalimantan, Indonesia, are typically male, married, 3–4 members in their household, and are Javanese.\n\nA small number of paddy households had the ability to buy hand tractor in the study areas (12.63%) (Table 1). There are significant differences the number of hand tractor owners among the eight paddy farm regions in East Kalimantan, Indonesia (χ2calculated 62.35 > χ2 table dk = 7; α = 0,05 14.1). Narayanamoorthy et al. (2014) found that the factors such as coverage of irrigation, yield enhancing inputs cost, land-labor ratio, and human labor use in man-hours have significanlty influence the use of machine labor in paddy cultivation.\n\nFarmers who did not own a hand tractor (87.37%) could rent from the owners of hand tractor who live in the same village or from nearby village (Table 2). There are significant differences the number of hand tractor renters among the eight paddy farm regions in East Kalimantan, Indonesia (χ2calculated 429.45 > χ2table dk = 7; α = 0,05 14.1). This was reasonable, because owning a hand tractor is very costly. Hand tractor prices ranged from IDR6,000,000.00 each-1 to IDR25,000,000.00 each-1.\n\nFarm size varies among paddy farming households in all regions (0.25-5.00 ha). Small land-holding farmers in the study areas did not have constraints to rent and use of hand tractors because the wetland fields for the most part are located in same area. However, Hristova & Maddock (1993) mentioned that land size could be a constraint in applying mechanized farming. The proportion of machine labour costs (11.13%) of total cost of cultivation of borewell irrigated paddy in Tumakuru District, India (Hamsa et al., 2017).\n\nThe tillage costs (Table 3) in Tenggarong Seberang and Muara Muntai were lower than those in Loa Janan. Tillage costs were same in other five regions. Limitations of the study, such as limited access to several study areas which more time was needed to collect data, influenced the diversity of respondents and data. There are significant differences the tillage costs among the eight paddy farm regions in East Kalimantan, Indonesia (χ2calculated 17.01 > χ2 table dk = 7; α = 0,05 14.1). The difference of tillage costs could be happened because of the difference of buying price of machine, operator wage, and machine maintenance cost.\n\nHand tractor usage is still recommended for the development of paddy farming as an important physical asset in paddy farming. The number of hand tractors in rural areas could be increased, either through purchase by paddy farmers or by grants from government, to decrease the tillage cost and production cost, thus increasing income of paddy farming and paddy farmers.\n\n\nConclusions\n\nThe number of paddy households as hand tractor owners and hand tractor renters in East Kalimantan in 2014 were 12.63% and 87.37%, respectively. The tillage cost was between IDR700,000.00 ha-1 and IDR1,000,000.00 ha-1. There are significant differences the number of hand tractor owners, the number of hand tractor renters, and the tillage costs among the eight paddy farm regions in East Kalimantan, Indonesia.\n\n\nData availability\n\nThe answers to the questionnaire, along with basic demographic information generated in this study are available on OSF. DOI: https://doi.org/10.17605/OSF.IO/C7EX9 (Karmini, 2018).\n\nThe questionnaire used in this study is available on OSF. DOI: https://doi.org/10.17605/OSF.IO/C7EX9 (Karmini, 2018).\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Grant information\n\nThe author declares that no grants were involved in supporting this work.\n\n\nReferences\n\nHamsa KR, Srikantha MPS, Gaddi GM: Comparison of cost and returns of major food crops under Central Dry Zone of Karnataka. IOSR-JAVS. 2017; 10(6): 21–26. Publisher Full Text\n\nHristova M, Maddock N: Private agriculture in Eastern Europe. Food Policy. 1993; 8(6): 459–462. Publisher Full Text\n\nKarmini: Comparison of Tillage Costs among Eight Paddy Farm Regions in East Kalimantan, Indonesia. OSF. Web, 2018.\n\nKarmini: Factors affecting paddy farm income in East Kalimantan, Indonesia. Biodiversitas. 2017; 18(1): 101–108. Publisher Full Text\n\nLarson DF, Plessmann F: Do farmers choose to be inefficient? Evidence from Bicol. J Dev Econ. 2009; 90(1): 24–32. Publisher Full Text\n\nNarayanamoorthy A, Bhattarai M, Suresh R, et al.: Farm mechanisation, MGNREGS and labour supply nexus: A state-wise panel data analysis on paddy and wheat crop. Ind Jn Of Agri Econ. 2014; 69(3): 320–335. Reference Source\n\nRea LM, Parker RA: Designing and Conducting Survey Research. A Comprehensive Guide. Jossey-Bass Publishers, San Fransisco, 1997. Reference Source" }	[ { "id": "42260", "date": "14 Jan 2019", "name": "Ahmad Shuib", "expertise": [ "Reviewer Expertise Natural Resource Economics", "Tourism Planning and Development" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIs the work clearly and accurately presented and does it cite the current literature?\nVery limited references and a few dated references.\n\nIs the study design appropriate and is the work technically sound?\nThis is a straight forward descriptive study using Chi-squared tests to determine significant differences. It would have been more informative if the author had included the derivation of the costs of the equipment and the tillage operation from the user’s actual expenditure.\n\nAre sufficient details of methods and analysis provided to allow replication by others?\nThe process of collecting data was sufficiently discussed although the determination of the sample size was not clearly explained. If the data collection was carried out by the researcher herself without any assistant, it must have taken a long time to get data from 380 respondents. Samples of 1 and 2 for Bontang North and Bontang South respectively was not justified.\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nFor a descriptive study to determine differences in usage of the machinery, the analysis is adequate. But for practical and policy decision purposes, the analysis may not provide sufficient evidence to help the relevant agencies to take the appropriate actions.\n\nAre all the source data underlying the results available to ensure full reproducibility?\nThe author has clearly discussed the sources of data used in the study.\n\nAre the conclusions drawn adequately supported by the results?\nThis is a simple descriptive study with straight forward conclusions. The analysis will be useful for further causal relationship analysis.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6557", "date": "07 May 2021", "name": "Karmini Karmini", "role": "Author Response", "response": "The authors revision for review on 14 January 2019 by Ahmad Shuib, University Putra Malaysia (UPM), Seri Kembangan, Malaysia. 1. Is the work clearly and accurately presented and does it cite the current literature? Reviewer: Very limited references and a few dated references. Authors: We added references of Pobhirun and Pinitsoontorn (2019), Statistics Indonesia (2008), Statistics East Kalimantan (2009), and Ogunbanwo et al. (2019) in section of References. 2. Is the study design appropriate and is the work technically sound? Reviewer: This is a straight forward descriptive study using Chi-squared tests to determine significant differences. Authors: We added methods of Descriptive statistics (to count the frequency, total, percentage, maximum, and minimum) and One Way Anova besides Chi Square One Sample (to test hypotheses) in section of Statistical analysis. Reviewer: It would have been more informative if the author had included the derivation of the costs of the equipment and the tillage operation from the user’s actual expenditure. Authors: We added information if the amount of hand tractor rental cost at the research location includes machine rental cost, machine maintenance cost, and operator wage in section of Tillage cost. 3. Are sufficient details of methods and analysis provided to allow replication by others? Reviewer: The process of collecting data was sufficiently discussed although the determination of the sample size was not clearly explained. Authors: We added method to determine the sample size in section of Subject recruitment. Reviewer: If the data collection was carried out by the researcher herself without any assistant, it must have taken a long time to get data from 380 respondents. Authors: We added second author and enumerators whereas the researchers employed the enumerators to help the interviews process in sections of Author and Subject recruitment. Reviewer: Samples of 1 and 2 for Bontang North and Bontang South respectively was not justified. Authors: The determination of number of samples for North Bontang (24 paddy households; 1 sample) and South Bontang (120 paddy households; 2 samples) was already appropriate based on proportional sampling method and already fulfilled the minimum sample size for population (380 samples) in section of Subject recruitment. 4. If applicable, is the statistical analysis and its interpretation appropriate? Reviewer: For a descriptive study to determine differences in usage of the machinery, the analysis is adequate. But for practical and policy decision purposes, the analysis may not provide sufficient evidence to help the relevant agencies to take the appropriate actions. Authors: We added recommendations if farm machinery are required to develop paddy farming also Bontang and Penajam Paser Utara are the recommended regions for the activities in relation to increase the ownership of hand tractors because have higher tillage costs rather than Kutai Kartanegara in section of Conclusions. 5. Are all the source data underlying the results available to ensure full reproducibility? Reviewer: The author has clearly discussed the sources of data used in the study. Authors: The questionnaire and data are already available in sections of Extended data and Underlying data 6. Are the conclusions drawn adequately supported by the results? Reviewer: This is a simple descriptive study with straight forward conclusions. Authors: We revised the results of hypotheses testing in sections of Conclusions and Abstract. Reviewer: The analysis will be useful for further causal relationship analysis. Authors: We added some factors caused differences the number of hand tractor owners, the number of hand tractor renters, and the tillage costs among some regions in East Kalimantan, Indonesia in section of Conclusions. Is the work clearly and accurately presented and does it cite the current literature? Reviewer: Partly Authors: Additional references in section of References. Is the study design appropriate and is the work tehnicaly sound? Reviewer: Yes Authors: Additional information in sections of Methods (Statistical analysis) and Results and discussion (Tillage cost). Are sufficient details of methods and analysis provided to allow replication by others? Reviewer: Partly Authors: Additional of method, second author, and enumerators in sections of Author and Methods (Subject recruitment). If applicable, is the statistical analysis and its interpretation appropriate? Reviewer: Yes Authors: Additional recommendations in section of Conclusions. Are all the source data underlying the results available to ensure full reproducibility? Reviewer: Partly Authors: The questionnaire and data already available in section of Data Availability (Underlying data and Extended data). Are the conclusions drawn adequately supported by the results? Reviewer: Yes Authors: Additional and revision of the analysis results in sections of Conclusions and Abstract." } ] }, { "id": "78026", "date": "11 Mar 2021", "name": "Ganganee Chandima Samaraweera", "expertise": [ "Reviewer Expertise Agricultural Economics", "Agricultural Marketing", "Consumer behavior" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nEven the author mentioned significant differences, p values are not given (specially in case of ownership and other variables, it says, (There are significant differences the number of hand tractor owners, the number of hand tractor renters, and the tillage costs among the eight paddy farm regions in East Kalimantan, Indonesia.). However, it didn't show clearly. The demographic status of the respondents is not mentioned, and the statistical analyses are not adequate as the author used only Chi-Square test. It is a serious issue as this is the only stat method used here. Better to expand the analytical tools as this cannot be generalized to the vast majority.\nMore recent literature is needed to be addressed and authors should mention the compatibility of their research findings with other relevant literature documented so far.\nIn the Methodology, it should be clearly mention the reason of selecting study areas, and the clear way of selecting the sample from the population\nHence, I will give my recommendation after adding those comments.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "6558", "date": "07 May 2021", "name": "Karmini Karmini", "role": "Author Response", "response": "The authors revision for review on 11 March 2021 by Ganganee Chandima Samaraweera, Department of Agricultural Economics, Faculty of Agriculture, University of Ruhuna, Kamburupitiya, Matara, Sri Lanka. Reviewer: Even the author mentioned significant differences, p values are not given (specially in case of ownership and other variables, it says, (There are significant differences the number of hand tractor owners, the number of hand tractor renters, and the tillage costs among the eight paddy farm regions in East Kalimantan, Indonesia.). However, it didn't show clearly. Authors: We added р-values in sections of Hand tractor ownership, Hand tractor renter, and Tillage cost. Reviewer: The demographic status of the respondents is not mentioned, Authors: We added Table 1 and information of respondents’ socioeconomic-demographic characteristics in section of Characteristics of respondents. Reviewer: and the statistical analyses are not adequate as the author used only Chi-Square test. It is a serious issue as this is the only stat method used here. Better to expand the analytical tools as this cannot be generalized to the vast majority. Authors: We added descriptive statistics to count the frequency, total, percentage, maximum, and minimum in section Statistical analysis. We revised Chi Square One Sample with One Way Anova to test hypothesis that there are no significant differences the tillage costs among Bontang, Kutai Kartanegara, and Penajam Paser Utara in sections of Statistical analysis and Tillage cost. Reviewer: More recent literature is needed to be addressed Authors: We added references of Pobhirun and Pinitsoontorn (2019), Statistics Indonesia (2008), Statistics East Kalimantan (2009), and Ogunbanwo et al. (2019) in section of References. Reviewer: and authors should mention the compatibility of their research findings with other relevant literature documented so far. Authors: Statistics Indonesia (2008) and Statistics East Kalimantan (2009) reported data of paddy farm income and paddy productivity in section Study area. Ogunbanwo et al. (2019) and Pobhirun and Pinitsoontorn (2019) used descriptive statistics and Chi Square test in their researches in section Statistical analysis. Reviewer: In the Methodology, it should be clearly mention the reason of selecting study areas, Authors: We added the reasons of selecting study areas associate with comparison of paddy farmer income and paddy productivity in provinces in Kalimantan in section Study area. Reviewer: and the clear way of selecting the sample from the population. Hence, I will give my recommendation after adding those comments. Authors: We added the random number method assigned every sample a number, then randomly picked samples by using a table of random numbers in section of Subject recruitment. Is the work clearly and accurately presented and does it cite the current literature? Reviewer: No Authors: Additional references in sections of Methods (Study area, Statistical analysis) and References. Is the study design appropriate and is the work tehnicaly sound? Reviewer: Partly Authors: Additional information in section of Methods (Study area and Subject recruitment). Are sufficient details of methods and analysis provided to allow replication by others? Reviewer: Partly Authors: Additional and revision of methods in sections of Methods (Statistical analysis) and Results and discussion (Tillage cost) If applicable, is the statistical analysis and its interpretation appropriate? Reviewer: Partly Authors: Additional the results of data analysis and information in section of Results and discussion (Characteristics of respondents, Hand tractor ownership, Hand tractor renter, and Tillage cost). Are all the source data underlying the results available to ensure full reproducibility? Reviewer: Partly Authors: The questionnaire and data already available in section of Data Availability (Underlying data and Extended data). Are the conclusions drawn adequately supported by the results? Reviewer: Partly Authors: Additional and revision of analysis results in sections of Conclusions and Abstract." } ] } ]	1	https://f1000research.com/articles/7-1951
https://f1000research.com/articles/9-1356/v1	20 Nov 20	{ "type": "Brief Report", "title": "Studying the prominence effect amid the COVID-19 crisis: implications for public health policy decision-making.", "authors": [ "Yossi Maaravi", "Ben Heller", "Ben Heller" ], "abstract": "The novel coronavirus disease 2019 (COVID-19) has brought with it crucial policy- and decision-making situations, especially when making judgments between economic and health concerns. One particularly relevant decision-making phenomenon is the prominence effect, where decision-makers base their decisions on the most prominent attribute of the object at hand (e.g., health concerns) rather than weigh all the attributes together. This bias diminishes when the decision-making mode inhibits heuristic processes. In this study, we tested the prominence of health vs. economic concerns across two decision-making modes - choice (prone to heuristics) and matching (mitigates heuristics) - during the peak of the COVID-19 in the UK using Tversky et al.’s classic experimental paradigm. We added to the classic experimental design a priming condition. Participants were presented with two casualty-minimization programs, differing in lives saved and costs: program X would save 100 lives at the cost of 55-million-pound sterling, whereas program Y would save 30 lives at the cost of 12-million-pound sterling. Half of the participants were required to choose between the programs (choice condition). The other half were not given the cost of program X and were asked to determine what the cost should be to make it as equally attractive as the program Y. Participants in both groups were primed for either: a) economic concerns; b) health concerns; or c) control (no priming). Results showed that in the choice condition, unless primed for economic concerns, health concerns are more prominent. In the matching condition, on the other hand, the prominence of health concerns did not affect decision-makers, as they all “preferred” the cheaper option. These results add further support to the practical relevance of using the proper decision-making modes in times of consequential crises where multiple concerns, interests, and parties are involved.", "keywords": [ "Covid-19", "Prominence effect", "Decision making", "Public policy" ], "content": "Introduction\n\nIn just a few months, coronavirus disease 2019 (COVID-19) has spread globally, taking lives and undermining economies1. Consequently, the World Health Organization announced a global emergency, and countries have taken numerous measures to fight it: isolation, quarantine, social distancing, etc.2.\n\nWhile these measures have clear health advantages, their effect on economies has been disastrous3 (e.g., the collapse of the traveling industry4). This tension between health and economic interests has led to numerous debates surrounding one question: how to judge different alternatives (e.g., two different programs to fight the pandemic) when they vary across more than one criterion (e.g., their death toll vs. their financial impact)?\n\nResearch in behavioral decision-making has long shown that decision-makers diverge significantly from the rational “homo-economicus” of classic economics5. Researchers suggested that uncertainty is a breeding ground for irrational or biased judgments that are based on emotions and heuristics6. Such patterns persist even during crucial decisions (e.g., health issues7) and those made by professionals (e.g., courtroom judges8).\n\nA relevant behavior is people’s tendency to overly focus on a single dominant attribute of objects they judge9. One particularly apt bias is the prominence effect - people’s tendency to focus on the most prominent attribute of judged objects instead of weighing all criteria10. Tversky et al. (1988) presented decision-makers a problem where health and economic considerations were mixed, a paradigm aptly suitable for research into the current situation. Using two decision modes (i.e., choice vs. matching), they showed that people tend to overly focus on the more salient attribute of casualties rather than costs. As detailed below, we used the same scenario amid the COVID-19 crisis with the following changes: the virus instead of traffic accidents and a town in the UK instead of Israel.\n\nWe hypothesized that unless decision mode (i.e., matching) or role manipulation deliberately made participants pay more attention to the economic criterion, they would overly emphasize casualties over costs.\n\n\nMethods\n\nParticipants from the UK (129 males and 291 females, all adults above 18 years of age) were recruited through the “Prolific” [22/4/2020] crowd-working platform and answered an online questionnaire for a pay. The only criterion for participation was that participants be above 18 years of age. Other than this requirement, participants were sampled in a “first come first serve” manner: whoever signed up for the study and was eligible to participate before reaching our target number of participants could answer the questionnaire. The participants were randomly assigned to the different experimental conditions. The study was conducted on the 22nd of April, 2020.\n\nParticipants read a scenario regarding the predicted number of COVID-19 related casualties (600) over the next year in a small town in the UK. They were then required to consider two programs (X vs. Y), described in terms of yearly costs (in millions of Pound Sterling £) and the number of casualties. Participants were asked to imagine themselves as either: Policymakers in the 1. Ministry of Treasury; 2. Ministry of Health; 3. Control group participants were not given a role.\n\nDecision measure. Half of the participants were required to choose between the two programs (choice condition): program X would lower casualties to 500 at the cost of 55-million-pound sterling, and program Y would lower casualties to 570 at the cost of 12-million-pound sterling. The output data for this condition was proportion of participants chosing program X vs. Y (See results section).\n\nThe other half received the same information except that program X’s price was missing. They were required to determine a cost that would make it as equally attractive as program Y (matching condition). To do so, they were first asked whether 55-million-pound sterling was the right number and to then type the appropriate cost. Missing casualty information was not used as another condition since Tversky et al. (1987) showed that the prominence effect occurred regardless of the dimension left missing. See the extended data sub-section at the end of this article for a complete description of the scenario and questionnaire11.\n\nAll statistical analyses were conducted using IBM SPSS Statistics for Windows, Version 22.0. In order to test for differences in proportion of participants who chose each program within each role (health, treasury, control) we conducted a binomial test, and to test for significant differences in these proportions between each role we conducted two chi-squared tests. Additionally, in order to test for differences in proportion of participants who thought that 55 million Pound Sterling was too high/low of a matching price within each role we conducted a binomial test. Finally, three single sample t-tests with 40 million Pound Sterling as a criterion were conducted in order determine whether participants within each role were willing to pay the corrected (triangulated) price.\n\nThis study was approved by the IRB at the Adelson School of Entrepreneurship, The Interdisciplinary Center, Herzliya (IRB confirmation no. 9). Informed consent was gathered from participants prior to their starting the study. They were given details as to the general topic of the study (decision making), the expected duration (5 minutes), and were assured that their answers were to be anonymously collected and no unsolicited use of their data would occur. Finally, they were informed that they may decide to not participate in the study after reading this information and could stop participating at any time if the study made them feel uncomfortable or for any reason.\n\n\nResults and discussion\n\nChoice condition. A binomial test indicated that the proportion of participants in the treasury group who chose program Y (69.4%) over program X (30.6%) was significantly greater than 50% (chance value), p = 0.001 (2-sided; see Figure 111). There was no significant difference in these proportions for participants in both the health (Y= 59.4%, X= 40.6%) and the control groups (Y= 52.9%, X= 47.1%). Additionally, Two chi-square tests examined the relationship between role and program-choice proportions. Whereas the proportions of health and control groups did not differ significantly, treasury and control groups were significantly different: χ2 (1, N = 142) = 4.11, p = 0.043 (see Figure 111).\n\nFigure depicting the percentage of participants who chose each program within each role. *p<0.001, †p<0.05.\n\nMatching condition. A binomial test indicated that the proportions of participants in the treasury (95%), health (97%), and control (98%) groups who thought that 55-million-pound sterling was too high a price were significantly greater than 50% (chance value), p < 0.001 (2-sided).\n\nTriangulating lives saved (in comparison to the 600 default) with the cost of program Y, resulted in the appropriate cost of program X being 40-million-pound sterling. Three single sample t-tests with 40 million pounds as the criterion indicated that participants in all roles thought it too high a price: treasury (M= 15.87, SD= 9.44), health (M= 16.43, SD= 7.65), and control (M= 16.19, SD= 9.73) groups: t (65)= -20.74, p< 0.001; t (67)= -25.38, p< 0.001; and t (60)= -19.10, p< 0.001 respectively. The same analysis using 20 as a stricter criterion yielded similar results in the treasury (t (65)= -3.54, p= 0.001), health (t (67)= -3.84, p< 0.001), and control (t (60)= -3.05, p= 0.003) groups.\n\nAt first glance, the results of the choice condition seem to diverge from Tversky et al.’s prominence effect. There, participants – who were not given any roles - had significantly preferred program X (higher costs, fewer casualties), which implied the prominence of saving lives over costs. But, although our results do not show a significant preference for program X in the control group, 50% of participants still chose it – despite its disproportionate cost to the lives-saved ratio. This finding, coinciding with the beginning of the lockdown in the UK (which made the economic issues much more salient and relevant), lends support to the claim that health issues were still the most prominent. To claim that the financial matters were more salient, we would see a significant preference for program Y. Nevertheless, only the treasury condition yielded this result. Coupled with the results of the matching condition below, it seems like an illustration of the prominence of health issues over economic issues in the choice condition.\n\nWhen matching, participants overwhelmingly preferred the more economical program. Indeed, they thought 55-million-pound sterling was too high of a matching price, and their proposed matching price was significantly lower than 40 and even 20. Comparing these results to the choice condition patterns suggests that “lives saved” was the more prominent attribute for participants who chose program X (approximately 50%). Only under the treasury condition did we see a difference in choice favoring the more economical program, demonstrating the prominence effect.\n\nTaken together, our results support Tversky et al.’s prominence effect study. They suggest that decision-makers should match the options instead of choosing between them in such decision-making situations.\n\n\nConclusion\n\nGiven the highly consequential decisions policymakers must make in times of crisis, it is crucial to understand which decision-making paradigms might lead to better, less biased decisions. The prominence of health over economic issues may lead to irrational differences between choosing versus matching. Thus, changing the decision-making paradigm from simply choosing alternatives to a matching paradigm can aid policymakers to better weigh all attributes.\n\n\nData availability\n\nOpen Science Framework: Covid-19 Prominence (F1000).\n\nhttps://doi.org/10.17605/OSF.IO/24Z3G11\n\nThis project contains the following underlying data:\n\nCovid-19_ Prominence (OSF).sav (The original dataset)\n\nCodebook.xlsx (key to understanding variables in dataset)\n\nOpen Science Framework: Covid-19 Prominence (F1000).\n\nhttps://doi.org/10.17605/OSF.IO/24Z3G11\n\nThis project contains the following extended data:\n\nCovid-19_ Prominence - Scenario.docx (The scenario and questionnaires used in the study)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nSohrabi C, Alsafi Z, O’Neill N, et al.: World Health Organization declares global emergency: A review of the 2019 novel coronavirus (COVID-19). Int J Surg. 2020; 76: 71–76. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilder-Smith A, Freedman DO: Isolation, quarantine, social distancing and community containment: pivotal role for old-style public health measures in the novel coronavirus (2019-nCoV) outbreak. J Travel Med. 2020; 27(2): taaa020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAtkeson A: What will be the economic impact of covid-19 in the us? rough estimates of disease scenarios. National Bureau of Economic Research. 2020. Publisher Full Text\n\nNicola M, Alsafi Z, Sohrabi C, et al.: The socio-economic implications of the coronavirus and COVID-19 pandemic: a review. Int J Surg. 2020; 78: 185–193. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNeale MA, Bazerman MH: Negotiator cognition and rationality: A behavioral decision theory perspective. Organ Behav Hum Decis Process. 1992; 51(2): 157–75. Publisher Full Text\n\nTversky A, Kahneman D: Judgment under uncertainty: Heuristics and biases. Science. 1974; 185(4157): 1124–31. PubMed Abstract \| Publisher Full Text\n\nSenay I, Kaphingst KA: Anchoring-and-adjustment bias in communication of disease risk. Med Decis Making. 2009; 29(2): 193–201. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDanziger S, Levav J, Avnaim-Pesso L: Extraneous factors in judicial decisions. Proc Natl Acad Sci U S A. 2011; 108(17): 6889–92. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMaaravi Y, Hameiri B: Deep pockets and poor results: The effect of wealth cues on first offers in negotiation. Group Decis Negot. 2019; 28(1): 43–62. Publisher Full Text\n\nTversky A, Sattath S, Slovic P: Contingent weighting in judgment and choice. Psychological review. 1988; 95(3): 371–384. Publisher Full Text\n\nHeller B: Covid-19 Prominence (F1000). 2020. http://www.doi.org/10.17605/OSF.IO/24Z3G" }	[ { "id": "79762", "date": "17 Mar 2021", "name": "Roshen Fernando", "expertise": [ "Reviewer Expertise Economic Policy", "Statistics", "Health Economics" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe brief report titled, assesses the implications of the prominence effect among the health and economic concerns in decision making in the public health sphere during a pandemic. The study evaluates the impact of two decision modes: choice vs matching. The authors use the classic experimental paradigm to base the survey and uses few statistical techniques to analyze the findings. The study concludes the importance of matching decision making mode in times of a pandemic.\nThe paper is well-written though quite brief. It is important to rethink whether the brief report could replace a more descriptive journal article format, especially when communicating finer points of the study. Otherwise, the value contribution to the body of knowledge could be minimal.\nThe study suffers primarily from two major limitations. Firstly, the authors seem to have confused the difference between financial and economic outcomes. What the authors involve in the study are financial outcomes, which are not in any way a substitute to the economic outcomes. In fact, healthy populations are central to an economy and during a pandemic, it is essential to restore confidence in the economy by controlling the infection spread as soon as possible. One lesson COVID-19 demonstrates is that the economic outcomes of countries which did not adopt lockdowns are not different to those who adopted lockdowns. These observations make the health vs economy a false debate. Unless the authors want to get into this debate, which clearly cannot be done using a brief report, the mentioning of economic outcomes should be replaced with financial outcomes, and the research should be framed accordingly.\nSecondly, the roles assigned to the participants, who are clearly submitting to the study due to a financial incentive, is not robust. I believe that the authors could figure out an alternative way to address the process of assigning roles. The financial figures presented also are arbitrary and it is questionable what the figures could have meant for the participants. Using something relatable, say 0.1% of GDP, could have made the process more robust.\nI suggest that the authors review their methodology and improve the results for it to be impactful.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [ { "c_id": "6614", "date": "07 May 2021", "name": "Yossi Maaravi", "role": "Author Response", "response": "Reviewer 1 The brief report titled, assesses the implications of the prominence effect among the health and economic concerns in decision making in the public health sphere during a pandemic. The study evaluates the impact of two decision modes: choice vs matching. The authors use the classic experimental paradigm to base the survey and uses few statistical techniques to analyze the findings. The study concludes the importance of matching decision making mode in times of a pandemic. The paper is well-written though quite brief. It is important to rethink whether the brief report could replace a more descriptive journal article format, especially when communicating finer points of the study. Otherwise, the value contribution to the body of knowledge could be minimal. Response: Thank you for taking the time to review our manuscript. We appreciate your comments and suggestions, and we are positive that thanks to them, our manuscript will be better suited for publication. The study suffers primarily from two major limitations. Firstly, the authors seem to have confused the difference between financial and economic outcomes. What the authors involve in the study are financial outcomes, which are not in any way a substitute to the economic outcomes. In fact, healthy populations are central to an economy and during a pandemic, it is essential to restore confidence in the economy by controlling the infection spread as soon as possible. One lesson COVID-19 demonstrates is that the economic outcomes of countries which did not adopt lockdowns are not different to those who adopted lockdowns. These observations make the health vs economy a false debate. Unless the authors want to get into this debate, which clearly cannot be done using a brief report, the mentioning of economic outcomes should be replaced with financial outcomes, and the research should be framed accordingly. Response: Thank you for this important feedback. We have come to agree with your claim that framing the manuscript as a “financial vs. health” trade-off would be more accurate given the relationship between economic prosperity and the population's health in question. We also agree that delving into the differences between financial and economic factors is beyond the scope of our brief report. We have thus modified our manuscript to reflect differences in decision-making modes regarding financial vs. health concerns. Secondly, the roles assigned to the participants, who are clearly submitting to the study due to a financial incentive, is not robust. I believe that the authors could figure out an alternative way to address the process of assigning roles. Response: We agree with this contention, yet we believe that the assignment to roles affected participants’ choice - as evidenced by our results - thus allowing us to derive insights from our study. Nevertheless, we have incorporated your feedback as a possible limitation in our manuscript. The financial figures presented also are arbitrary and it is questionable what the figures could have meant for the participants. Using something relatable, say 0.1% of GDP, could have made the process more robust. Response: While we agree that these figures may not be relatable and may not mean much to the participants, we used the same figures that Tversky, Sattath, and Slovic used in their seminal article “Contingent Weighting in Judgement and Choice” (1987). We wished to conform as much as possible to their original paradigm while only changing what was necessary to answer our research questions. Nevertheless, we have incorporated your feedback as a limitation in our manuscript." } ] }, { "id": "81287", "date": "15 Apr 2021", "name": "Peterson K Ozili", "expertise": [ "Reviewer Expertise Economic policy making" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article examines an important issue about the COVID-19 pandemic – how people choose between casualties (a health consideration) over costs (an economic consideration). The authors assess whether people would choose casualty over cost, and whether they do so when their attention is drawn to more specific economic realities. The method used conform to ethics such as using adult participants, transparent payment for participation, informed consent of participants, equal amount of time for experiment, etc.\nAlthough the number of females exceed the male participants, I don’t think the skewed gender size matters as it may not affect the result because the reaction of males and females to unpleasant experiences is relatively the same in most cases. The research instrument and design is appropriate, and more interestingly, the findings support the prominence effect of Tversky et al. (1988)1.\nIn the conclusion section, please suggest some areas for future research. In section 1, no citations were provided to support the claim about the conflcting health tension (e.g. to save people) and the economic tension (to save the economy). The authors can try to expand on why the economic cost matters during the pandemic. To address this, I suggest a few papers that could help the authors in the regard, especially, McKibbin & Fernando (2021)2, and Ozili and Arun (2020)3. These two articles clearly breakdown the economic costs of the pandemic on individuals, firms and countries. The second paper is one of my articles on the economic consequences of COVID-19.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6615", "date": "07 May 2021", "name": "Yossi Maaravi", "role": "Author Response", "response": "Reviewer 2 The article examines an important issue about the COVID-19 pandemic – how people choose between casualties (a health consideration) over costs (an economic consideration). The authors assess whether people would choose casualty over cost, and whether they do so when their attention is drawn to more specific economic realities. The method used conform to ethics such as using adult participants, transparent payment for participation, informed consent of participants, equal amount of time for experiment, etc. Although the number of females exceed the male participants, I don’t think the skewed gender size matters as it may not affect the result because the reaction of males and females to unpleasant experiences is relatively the same in most cases. The research instrument and design is appropriate, and more interestingly, the findings support the prominence effect of Tversky et al. (1988)1. Response: Thank you for taking the time to review our manuscript. We appreciate your comments and suggestions, and we are positive that thanks to them, our manuscript will be better suited for publication. In the conclusion section, please suggest some areas for future research. Response: Thank you for this suggestion. We also believe that this study offers multiple opportunities for future research endeavors. Therefore, we have added a paragraph discussing the limitations of our study and possible future research directions. In section 1, no citations were provided to support the claim about the conflcting health tension (e.g. to save people) and the economic tension (to save the economy). The authors can try to expand on why the economic cost matters during the pandemic. To address this, I suggest a few papers that could help the authors in the regard, especially, McKibbin & Fernando (2021)2, and Ozili and Arun (2020)3. These two articles clearly breakdown the economic costs of the pandemic on individuals, firms and countries. The second paper is one of my articles on the economic consequences of COVID-19. Response: Thank you for this remark and your recommendations. We agree that this point could have been made in a more comprehensive fashion, and have thus elaborated on the health-economy tradeoff. Your suggested papers have proven to be very informative and useful in this regard." } ] } ]	1	https://f1000research.com/articles/9-1356
https://f1000research.com/articles/10-357/v1	07 May 21	{ "type": "Research Article", "title": "Quality of solo ambulance care by physician assistants versus ambulance nurses for non-conveyed patients in the Netherlands: An observational study", "authors": [ "Lilian Vloet", "Daniël Winterink", "Aico Gerritsen", "Wim Heutz", "Thijs van Zonneveld", "Sivera Berben", "Remco Ebben", "Lilian Vloet", "Daniël Winterink", "Aico Gerritsen", "Wim Heutz", "Thijs van Zonneveld", "Sivera Berben" ], "abstract": "Background The aim of this study was to compare the ambulance care process, follow-up care and patient experience between physician assistants and ambulance nurses operating as solo ambulance care providers, for a non-conveyed patient population.\nMethods An observational design was used. Characteristics of patients and events and the care process were retrieved from the ambulance registration database. Data on follow-up care and patient experience were collected through questionnaires.\nResults Of the included solo ambulance events, 49/379 (12.9%) were performed by physician assistants, 330/379 (87.1%) were performed by ambulance nurses. For initial complaints and the on-scene diagnoses there were no significant differences between the physician assistants and ambulance nurses. 90/165 (54.5%) of the patients requested follow-up care after being non-conveyed with no significant association between the PA and ambulance nurse group (p=.293). For type of follow-up care, 91.9% of the follow-up emergency care requests in this study came from patients treated by an ambulance nurse. There were no significant differences in patients’ experience for clinician attitude and behavior, treatment, and communication between physician assistants and ambulance nurses, although pain management and explanation about the non-conveyance decision could be improved\nConclusions Besides small statistical but not clinically significant differences, this study indicated comparable solo ambulance care provided by a physician assistant or an ambulance nurse concerning the care process, follow-up care and patient experience. Patients treated by physician assistants seek less follow-up emergency care after non-conveyance, compared to ambulance nurses. Overall, patients experienced good attitude and behavior, treatment, and communication of the solo ambulance professional. Future well powered studies to gain insight in effects of PAs in ambulance care are needed, as well as studies in which PAs apply all additional skills they are licensed to.", "keywords": [ "Emergency Medical Services [MeSH]", "Quality of Healthcare [MeSH]", "Physician Assistants [MeSH]", "non-conveyance" ], "content": "Introduction\n\nEmergency medical services (EMS) face a challenging environment with an increasing number of ambulance deployments1,2. Reasons for this increase are a growing population with more complex healthcare problems and comorbidities, repeated requests for ambulance care, and the request of ambulance care for primary care problems3–6. Within this increasing demand, there is also a growing population that receives ambulance care without conveyance to another healthcare facility. Non-conveyance is defined as an ambulance dispatched with on-scene assessment and treatment, but without conveyance to a hospital7,8. The non-conveyance decision can be initiated by the ambulance professional (sometimes after consultation with a general practitioner or medical specialist) or the patient and/or their relatives. For general patient populations the non-conveyance proportion ranges from 3.7–93.7%9. Although the proportion of non-conveyance is increasing, ambulance care professionals experience the conveyance decision making process as difficult, challenging and often unsupported by guidelines10,11. Patients are mostly satisfied with provided care in non-conveyance situations, but non-conveyance evokes fear, shame, and a need for reassurance12,13.\n\nTo meet the increased demand and patient complexity while securing patient safety and effectiveness in ambulance care, stratification of ambulance care using different types of ambulance care professionals is needed. Therefore, physician assistants (PA’s) were introduced in ambulance care in the Netherlands. Primary rationale for the introduction is to provide efficient and adequate care by different types of ambulance care professionals to meet the growing demand. Secondarily, to retain ambulance nurses EMSs want to offer ambulance nurses a career perspective at a master’s level, such as the nurse practitioner or physician assistant roles14.\n\nDespite the introduction of the PA in ambulance care, little is known about the effects. A recent systematic review showed limited available evidence on the introduction of master’s level educated professionals in ambulance care, with lacking evidence on patient outcomes15. This underpins the need for further research on effectiveness, efficiency, patient safety, and accessibility of the introduction of PAs in ambulance care. Within the chain of emergency care, a systematic review showed that PAs in the emergency department setting are reliable in assessment of medical complaints and performing procedures, and that PAs are accepted by patients and ED staff16.\n\nAs the effects of PAs working in prehospital ambulance care are currently unclear, the aim of this study was to compare the ambulance care process, follow-up care, and patient experience between physician assistants and ambulance nurses operating as solo ambulance care providers, for a non-conveyed patient population.\n\n\nMethods\n\nThis study had an observational design and is reported using the STROBE-statement17. Ethical approval was obtained from the Ethical Research Committee of the HAN University of Applied Sciences (see ethical statement below).\n\nAmbulance care in the Netherlands is dispatched through the dispatch center and can be requested via the national emergency number or by healthcare professionals (such as general practitioners). After triage, ambulance care can be dispatched with urgency level A1 (arrival <15 minutes), A2 (arrival <30 minutes, and B (planned ambulance care). The dispatch center can dispatch a fully equipped ambulance with conveyance facilities (one driver and one ambulance nurse) or a solo ambulance unit. The non-nurse driver at the fully equipped ambulance has a supporting role to the ambulance nurse during the care and transfer process. A solo ambulance unit is an ambulance vehicle with basic and advanced life support equipment but without conveyance facility18. A solo ambulance unit can be staffed by one ambulance care professional, either an ambulance nurse, physician assistant or nurse practitioner.\n\nTraditionally, ambulances in the Netherlands are staffed by ambulance nurses. Ambulance nurses in the Netherlands are registered nurses (RN) with additional education and working experience in anesthesia, Intensive Care (IC) or emergency department care (ED), before entering the 9-month ambulance training program. After graduation from the ambulance training program, ambulance nurses have functional autonomy within the framework of their national EMS protocols19,20. In comparison to ambulance nurses, The PA holds a master’s degree in the medical domain, the 30 month educational program has theoretical and clinical phases, with a strong focus on diagnostic skills, such as organ tract system examination14,21. PAs have advanced diagnostic skills at master level and they are allowed by law to independently diagnose and treat patients beyond the national EMS protocol. Furthermore, they can perform autonomously advanced medical procedures, such as surgical procedures, endoscopy, punctures, elective cardioversions, and medication prescription.\n\nThis study was performed an urban EMS region in the Netherlands. This EMS uses a combination of advance life support and basic life support ambulance units, dispatch in this region is guided by the Dutch Triage Standard22. The solo ambulance care unit in this EMS is solely dispatched between 07:00h-23.00h as historical data showed non-conveyance occurs more in urban areas and mostly during these hours. The solo ambulance care provider at this EMS is either an ambulance nurse or a PA. For this EMS 12 ambulance nurses and 5 PAs are eligible to staff the solo ambulance. All PAs worked as ambulance nurses before they entered the PA educational program.\n\nIn the Netherlands, ambulance nurses and PAs are eligible to apply for the solo ambulance care selection procedure when they have a minimum of 3 years working experience as ambulance nurse and hold an Advanced Medical Life Support certificate. The selection procedure consists of an interview assessment, medical assessment, and driving assessment. After passing this selection procedure, candidates undertake an additional 5-day driving training course with a theoretical and practical exam, and a 1-day training program aimed at (a) on-scene safety management, (b) incident management, (c) starting a resuscitation on your own, and (d) mass casualty incident coordination. Finally, they follow a 5-day traineeship with an experienced solo ambulance care professional.\n\nIn the period January-July 2019 this EMS region performed 40,910 ambulance dispatches, of which 1,549 were performed by a solo ambulance unit. Each dispatch has a unique identification number that is automatically generated. From the EMS we received the unique identification numbers of all ambulance dispatches with patient contact that ended in non-conveyance, except when (a) the ambulance run was cancelled by the dispatch center in case the ambulance was no longer needed, (b) the patient was conveyed by a regular ambulance after solo ambulance attendance and assessment, (c) the solo ambulance assisted another regular ambulance, (d) the patient deceased, (e) the patient refused care, (f) the solo ambulance run performed was for stand-by purposes, and (g) the patients’ home address was not available in the ambulance registration database.\n\nData was collected from 1) the ambulance registration database and 2) by questionnaires.\n\nThe ambulance registration database is an internal EMS database in which they register all data taken on an ambulance run. It can only be accessed by EMS managers and data managers, and on request for research purposes.\n\nCharacteristics of the patient (age, sex, time and day of ambulance request, and vital functions/observation scales), and the care process (EMS dispatch complaint, and initial on-scene diagnosis) were retrieved from the ambulance registration system. The initial complaint and initial on-scene diagnoses were categorized into one the of the 23 chapters of the International Statistical Classification of Diseases and Related Health Problems 10th revision (ICD-10)23. Categorization was performed by two independent researchers (DW, TvZ). In case of doubt a third researcher was consulted (RE).\n\nTo collect data on follow-up care and patient experience a questionnaire was developed. The first part of the questionnaire contained six questions about follow-up care after non-conveyance (repeated access to healthcare, relation between first EMS contact and repeated healthcare contact, reasons for repeated access to healthcare, and whether the patient received a prescription during a second healthcare contact). These questions were based on recent literature on non-conveyance9. The second part of the questionnaire focused on patient experience and was based on the validated Consumer Quality Index Emergency Ambulance Care (CQI-index)24. This CQI-index is a 48-item validated questionnaire to measure patient experience on seven scales: emergency number and dispatch center, attitude and behavior of the ambulance professional, treatment by ambulance professional, communication by ambulance professional, non-conveyance, transportation, and emergency department. The CQI-index was validated through factor analysis, assessing Cronbach’s alpha, and face validity, and the scales showed moderate to good reliability (Cronbach’s alpha 0,65-0,80)25. As this study focuses on on-scene and follow-up care by a solo ambulance care unit, we only used the 13 items from the four scales attitude and behavior, treatment, communication and non-conveyance. Patients could answer on a 4-point scale (‘no, not at all’, ‘yes, a little’, ‘yes, for the most part’, ‘yes, totally’), with an additional option for ‘can’t remember’ or ‘not applicable’. An information letter, the questionnaire, and a return envelope were sent to the included patients. The information letter contained information about the aim, anonymity, privacy, data management, informed consent, and contact information. The information letter also stated that if the patient returned the questionnaire, this was interpreted as informed consent to participate. Reminders, including the questionnaire and a return envelope, were sent after three weeks. Data from the ambulance registration database and questionnaires were linked on patient level by using the unique identification number that is automatically generated at ambulance dispatch. This linkage was performed by the EMS, the researchers only had access to an anonymized database. All variables from the ambulance registration database and questionnaire are displayed in Extended data.\n\nResults are presented using descriptive statistics. To determine effects between the PA and ambulance nurse groups, Chi-square tests, Fisher’s exact tests, and t-tests were performed. Statistical significance was defined as a p-value < 0.05. Missing data were at random, all statistical tests were performed on available data, no imputation occurred. Data were analyzed using SPSS version 25.0.\n\n\nResults\n\nOf the 1,549 solo ambulance events, 379 (24.5%) met inclusion criteria (Figure 1). Main reasons for exclusion were the patient being conveyed by an ambulance after solo ambulance assessment, and a standby ambulance run. Of the included solo ambulance events, 49/379 (12.9%) were performed by PAs, 330/379 (87.1%) were performed by ambulance nurses.\n\nThere were more female patients non-conveyed by PAs, compared to ambulance nurses (p=.042) (Table 1). The average age of the whole group was 43.1 years (SD 24.8) with no differences between the PA and the ambulance nurses’ group (43.2 years vs. 43 years, p=.952). 41.4% of the ambulance was requested between 17.00h-23.00h, there was no difference between PAs and ambulance nurses (p=.233). For the distribution of solo ambulance care runs across days of the week there was an overall statistical difference between the PA and the ambulance nurses’ groups, with no solo ambulance runs by PAs on Wednesdays (X2 = 21.605, p=.001).\n\nSIGNIFICANT AT P<0.05\n\n# X2-TEST\n\n† FISHER’S EXACT TEST\n\n‡‡ T-TEST\n\nThe initial complaints and on-scene diagnoses are described in Table 2. The initial complaints (X2 = 7,354, p=.807) and on-scene diagnoses (X2 = 7,711, p=.773) were similar in the PA and ambulance nurse groups. The top three initial complaints during EMS dispatch could be classified into injury, poisoning and certain other consequences of external causes; other/non classifiable; and mental, behavioral and neurodevelopmental disorders. This top three was equal for the PA and ambulance nurse group. For 278/379 (73.4%) of the patients on-scene diagnosis were available. The three most frequent diagnoses for the total group and ambulance nurses’ group could be classified into injury, poisoning and certain other consequences of external causes; other/non classifiable; and diseases of the circulatory system. For the PA group, certain infectious and parasitic diseases, diseases of the nervous system, and diseases of the circulatory system, completed the top three. Data on vital functions/observation scales largely showed comparable populations for PAs and ambulance nurses, with exception of insufficiency of breathing (0.3% vs. 5%, p=.038) (Table 3).\n\n†FISHER’S EXACT TEST\n\nSIGNIFICANT AT P<0.05\n\n#X2-TEST\n\n†FISHER’S EXACT TEST\n\nQuestionnaires to measure follow-up care and patient experience were returned by 165/379 respondents (43.5%), of which 18/165 (10.9%) were treated by PAs and 147/165 (89.1%) were treated by ambulance nurses (Figure 1).\n\nA total of 90/165 (54.5%) of the patients requested follow-up care after being non-conveyed by the solo ambulance care unit (Table 4). There was no significant association between follow-up care requests between the PA and ambulance nurse group (X2 = 5,328, p=.293), but 91.9% of the follow-up emergency care requests in this study came from patients treated by an ambulance nurse. Of the patients that requested follow-up care, 52/90 (57.8%) requested this follow-up care within 12 hours after being non-conveyed. Adding the numbers of patients who received follow up care within 12 hours, 24 hours and 48 hours’ time intervals, a total of 74/90 (82.2%) of the patient requested follow-up care within 48 hours. There was no significant association between the timeframe of the follow-up care request and the educational level of the ambulance care professional. The degree of suffering from health after being non-conveyed was higher in the ambulance nurse group, compared to the PA-group (X2. = 6,976, p=.047).\n\n*SIGNIFICANT AT P<0.05\n\n# X2-TEST\n\n† FISHER’S EXACT TEST\n\nThere were no significant differences in how patients experienced attitude and behavior, treatment, communication and the non-conveyance decision between PAs and ambulance nurses (Extended data Appendix 2), however the lower scores (‘not at all’ and ‘yes a little’) were only reported for ambulance nurses. For attitude and behavior, patients experienced the ambulance professional as polite, felt taken seriously, and felt that the ambulance professional took enough time. For treatment, patients felt reassured, trusted the ambulance professional, felt that the ambulance professional took firm action, and that the ambulance professional payed sufficient attention to other people present (family, friends, bystanders). 8.6% of the patients experienced that their pain was not or poorly managed by the ambulance care professional. For communication, patients experienced that the ambulance professional provided enough (understandable) information about what he was doing, and patients experienced the opportunity to ask questions. However, concerning the non-conveyance decision, 9.9% of the patients experienced that the reason why they were not being conveyed was not or a little explained by the ambulance professional.\n\n\nDiscussion\n\nThe aim of this study was to compare the ambulance care process, follow-up care, and patient experience between physician assistants and ambulance nurses operating as solo ambulance care providers, for a non-conveyed patient population. It showed parity between ambulance care provided by physician assistants and ambulance nurses, but showed differences in gender, days of the week, insufficiency of breathing, and degree of suffering. Also, the study indicated that pain management and explanation about the non-conveyance decision could be improved for non-conveyed patients.\n\nThe population in this study was comparable to other studies on non-conveyance with regard to distribution of gender and week days, although the patients in our study were slightly younger14,19,26,27. The differences in distribution across the week could be explained by workforce scheduling by the EMS. The initial complaints during dispatch did not differ between the PA and ambulance nurse group, and are partly comparable with a previous study reporting fall/fall of height, traffic accidents, overdoses/poisoning as EMS initial complaints26. There were no significant differences between the PA and ambulance nurse groups for on-scene diagnosis, and these diagnoses were comparable with previous studies on non-conveyance14,19,26,27. Our results indicate that PAs and ambulance nurses come to comparable diagnoses after being dispatched to a group with homogenous initial complaints during dispatch. The statistically significant difference found for insufficient breathing might be due to more advanced clinical reasoning skills of the PA compared to the ambulance nurses.\n\nIn this study 54.5% of the patients requested follow-up care after being non-conveyed by the solo ambulance care provider, with no significant differences in follow-up rates between self-referred or professional referred between the patients seen by the PA or an ambulance nurse. Although there was no significant association between type of follow-up care and educational level of the ambulance care professional, 91.9% of the follow-up emergency care requests in this study came from patients treated by an ambulance nurse, although another study showed comparable follow-up emergency care requests by PAs and ambulance nurses14. A possible explanation for this difference might be that a PA has more focus on the organ tract system examination due to their different educational background, resulting in more accurate diagnosis or treatment. Furthermore, in our study 57.8% requested follow-up care within 12 hours, and 82.2% requested follow-up care within 48 hours after being non-conveyed. To measure quality of non-conveyance within the EMS system, a follow-up indicator to measure the proportion of new healthcare contacts with time intervals at 24h, 48h, 72h and one week would be required. Furthermore, differentiation can be made for follow-up contacts as advised by the ambulance professional, or for new or recurrent complaints.\n\nOverall patient experiences of attitude and behavior, treatment, communication, and the non-conveyance decision by the solo ambulance professional were good, which is comparable with the literature13. From the patient perspective, there was room for improvement in the management of pain and the explanation of the non-conveyance decision. The suboptimal management of pain in EMS context has been reported previously28. In this study, it might also explain the differences in degree of suffering between patients treated by an ambulance nurse compared to the PA. Possibly, the explanation of the non-conveyance decision to the patient might be suboptimal, as the decision is perceived as complex, and ambulance professionals experience a lack of education regarding non-conveyance assessments and decisions10,11. Also, patients experience fear as prominent emotion, which might make it more difficult for them to understand and remember a provided explanation of the non-conveyance decision12. As the proportion of non-conveyance in EMS systems is substantial and increasing, it is important to improve communication about the non-conveyance decision. This urges the need to develop educational programs not only aimed at making safe non-conveyance decisions, but also teaching how to communicate this with the patient and significant others through the concept of shared decision making. Furthermore, patients often expect conveyance while requesting ambulance care10. This addresses the need to inform patients and manage their expectations when calling an ambulance.\n\nBesides the small statistical but not clinically significant differences in gender, days of the week, insufficiency of breathing, and degree of suffering, the care process, follow-up care, and patient experience were comparable between the ambulance nurse and PA. This might possibly be explained by the high educational level of ambulance nurses in the Netherlands, and the lack of clear task descriptions and national EMS standards for PAs in ambulance care. Therefore, PAs are not fully able to apply all medical and diagnostic skills (like drug prescription) during their professional practice, which might make a difference in prehospital care. Additionally, the effect of the PA on quality of ambulance care should be further assessed. Future research on the impact of the PA within ambulance care should be assessed by using the six dimensions of quality of healthcare: (1) effectiveness, (2) efficiency, (3) patient safety, (4) accessibility, (5) timeliness and (6) target population directed15. The effects on these dimensions should be measured in all phases of the ambulance process, from initial call and dispatch triage, to handover or referral. Also, the implementation of the PA should be investigated, by gaining in-depth insight in factors influencing this implementation process from the perspective of the patient, the professional, the organization, and chain of emergency care.\n\nDespite the fact that this study is one of the first reporting on the quality of care of PAs in ambulance care, this study had several limitations. The first limitation is the partial use of retrospective data and the relatively small sample size for the patients seen by a PA, which means that the results could be biased by a lack of power. For future research to test differences in follow-up care after non-conveyance, we calculated a sample size estimation in order to achieve more statistical power. Based on a 95% confidence interval, a non-conveyed population size of 10,000–15,00027 and a 5% margin of error, a sample size of 370–375 would be advisable for future studies. Secondly, the questionnaire used was developed based on a validated instrument and literature, but was not additionally validated for this study. Thirdly, due to random suboptimal registration we had missing data on all variables, especially vital functions/observation scales. Missing data in non-conveyance studies has been described earlier19. Finally, the Netherlands has a specific EMS-system and this might limit generalizability to other healthcare systems.\n\n\nConclusion\n\nBesides small statistical but not clinically significant differences, this study indicated similarity in solo ambulance care provided by a physician assistant or an ambulance nurse concerning the care process, follow-up care, and patient experience. Regarding the type of follow-up care, patients treated by a PA seek less emergency care after being non-conveyed, compared to an ambulance nurse. Overall, patients experienced good attitude and behavior, treatment, and communication from the solo ambulance professional, with no differences between the PAs and ambulance nurses. From the patient’s perspective, pain management and explanation about the non-conveyance decision could be improved. Future well-powered studies to gain insight in effects of PAs in ambulance care are needed, as well as studies in which PAs apply all additional skills they are licensed to.\n\n\nData availability statement\n\nDANS-EASY: Underlying data for ‘Quality of solo ambulance care by physician assistants versus ambulance nurses for non-conveyed patients in the Netherlands: An observational study’ https://doi.org/10.17026/dans-zqw-qbqp29.\n\nThis project contains the following underlying data:\n\nAnonomised dataset (title: 20210407 Anonymized dataset.sav)\n\nSTROBE-statement\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Universal (CC0 1.0) Public Domain Dedication).\n\nDANS-EASY: Underlying data for ‘Quality of solo ambulance care by physician assistants versus ambulance nurses for non-conveyed patients in the Netherlands: An observational study’ https://doi.org/10.17026/dans-zqw-qbqp29.\n\nThis project contains the following extended data:\n\nAppendix 1\n\nAppendix 2\n\nExtended data are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Universal (CC0 1.0) Public Domain Dedication).\n\n\nEthical approval\n\nThe Ethical Research Committee of the HAN University of Applied Sciences critically appraised the research protocol and concluded that the study complied with the criteria of the Declaration of Helsinki on Ethical Principles for Medical Research Involving Human Subjects, applicable national laws (like the General Data Protection Regulation), and the Dutch code of conduct for Research Integrity (reference Ethical Research Committee HAN University of Applied Sciences: 241.02/21).\n\n\nConsent statement\n\nPatient were given an information letter alongside their questionnaire, which stated that if the patient returned the questionnaire, this was interpreted as informed consent to participate. In this way written informed consent for publication of the patients’ details was obtained from the patients.", "appendix": "Acknowledgements\n\nThe authors would like to thank Steven Teerenstra PhD. for his statistical advise during data-analysis, and Rianne van Boekel PhD and Paul JT Rood RN PhD for their feedback during the writing process.\n\n\nReferences\n\nLowthian JA, Cameron PA, Stoelwinder JU, et al.: Increasing utilisation of emergency ambulances. Aust Health Rev. 2011; 35(1): 63–69. PubMed Abstract \| Publisher Full Text\n\nChristensen EF, Larsen TM, Jensen FB, et al.: Diagnosis and mortality in prehospital emergency patients transported to hospital: a population-based and registry-based cohort study. BMJ Open. 2016; 6(7): e011558. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEdwards MJ, Bassett G, Sinden L, et al.: Frequent callers to the ambulance service: patient profiling and impact of case management on patient utilisation of the ambulance service. Emerg Med J. 2015; 32(5): 392–396. PubMed Abstract \| Publisher Full Text\n\nSøvsø¸ MB, Kløjgaard TA, Hansen PA, et al.: Repeated ambulance use is associated with chronic diseases - a population-based historic cohort study of patients' symptoms and diagnoses. Scand J Trauma Resusc Emerg Med. 2019; 27(1): 46. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBooker MJ, Shaw ARG, Purdy S: Why do patients with 'primary care sensitive' problems access ambulance services? A systematic mapping review of the literature. BMJ Open. 2015; 5(5): e007726. Publisher Full Text\n\nJones CMC, Wasserman EB, Li T, et al.: The Effect of Older Age on EMS Use for Transportation to an Emergency Department. Prehosp Disaster Med. 2017; 32(3): 261–268. PubMed Abstract \| Publisher Full Text\n\nCone DC, Kim DT, Davidson SJ: Patient-initiated refusals of prehospital care: ambulance call report documentation, patient outcome, and on-line medical command. Prehosp Disaster Med. 1995; 10(1): 3–9. PubMed Abstract \| Publisher Full Text\n\nLarsen T, Bendtsen M, Søvsø M, et al.: Treat-and-release EMS patients in the north denmark region: identification and vital signs. BMJ Open. 2017; 7: A13. Publisher Full Text\n\nEbben RHA, Vloet LCM, Speijers RF, et al.: A patient-safety and professional perspective on non-conveyance in ambulance care: a systematic review. Scand J Trauma Resusc Emerg Med. 2017; 25(1): 71. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLederman J, Löfvenmark C, Djärv T, et al.: Assessing non-conveyed patients in the ambulance service: a phenomenological interview study with Swedish ambulance clinicians. BMJ Open. 2019; 9(9): e030203. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHöglund E, Schröder A, Möller M, et al.: The ambulance nurse experiences of non-conveying patients. J Clin Nurs. 2019; 28(1–2): 235–244. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvan Doorn SCM, Verhalle RC, Ebben RHA, et al.: The experience of non-conveyance following emergency medical service triage from the perspective of patients and their relatives: A qualitative study. Int Emerg Nurs. 2021; 54: 100952. PubMed Abstract \| Publisher Full Text\n\nKing R, Oprescu F, Lord B, et al.: Patient experience of non-conveyance following emergency ambulance service response: A scoping review of the literature. Australas Emerg Care. 2020; S2588-994X(20)30083-X. PubMed Abstract \| Publisher Full Text\n\nBloemhoff A, Schoonhoven L, de Kreek AJ, et al.: Solo emergency care by a physician assistant versus an ambulance nurse: a cross-sectional document study. Scand J Trauma Resusc Emerg Med. 2016; 24: 86. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvan Vliet R, Ebben R, Diets N, et al.: Nurse practitioners and physician assistants working in ambulance care: A systematic review [version 1; peer review: 1 approved, 2 approved with reservations]. F1000Res. 2020; 9: 1182. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDoan Q, Sabhaney V, Kissoon N, et al.: A systematic review: The role and impact of the physician assistant in the emergency department. Emerg Med Australas. 2011; 23(1): 7–15. PubMed Abstract \| Publisher Full Text\n\nvon Elm E, Altman DG, Egger M, et al.: Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement: guidelines for reporting observational studies. BMJ. 2007; 335(7624): 806–808. Publisher Full Text\n\nCarlström E, Fredén L: The first single responders in Sweden - Evaluation of a pre-hospital single staffed unit. Int Emerg Nurs. 2017; 32: 15–19. PubMed Abstract \| Publisher Full Text\n\nBreeman W, Poublon NA, Verhofstad MHJ, et al.: Safety of on-scene medical care by EMS nurses in non-transported patients: a prospective, observational study. Scand J Trauma Resusc Emerg Med. 2018; 26(1): 79. Publisher Full Text\n\nEbben RH, Vloet LC, Schalk DM, et al.: An Exploration of Factors Influencing Ambulance and Emergency Nurses' Protocol Adherence in the Netherlands. J Emerg Nurs. 2014; 40(2): 124–30. PubMed Abstract \| Publisher Full Text\n\nLovink MH, Persoon A, van Vught AJAH, et al.: Substituting physicians with nurse practitioners, physician assistants or nurses in nursing homes: protocol for a realist evaluation case study. BMJ Open. 2017; 7(6): e015134. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDrijver CR: [Use the Netherlands Triage Standard for children]. Ned Tijdschr Geneeskd. 2015; 159: A8330. PubMed Abstract\n\nWHO Library Cataloguing-in-Publication Data:International statistical classification of diseases and related health problems. - 10th revision, edition 2010. 2011. Reference Source\n\nVan de Ven D, Bos N, De Boer D: Kwaliteit van ambulancezorg vanuit het perspectief van cliënten: ervaringen van cliënten met de ambulancezorg gemeten met de CQ-indexen planbare en spoedeisende ambulancezorg. 2017. Reference Source\n\nKrol M, Sixma H, Plass AM: CQI Spoedeisende Ambulancezorg: actualisatie en bepaling van het discriminerend vermogen. 2013: 1. Reference Source\n\nEbben RHA, Castelijns M, Frenken J, et al.: Characteristics of non-conveyance ambulance runs: A retrospective study in the Netherlands. World J Emerg Med. 2019; 10(4): 239–243. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVloet LCM, de Kreek A, van der Linden EMC, et al.: A retrospective comparison between non-conveyed and conveyed patients in ambulance care. Scand J Trauma Resusc Emerg Med. 2018; 26(1): 91. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBerben SA, Schoonhoven L, Meijs TH, et al.: Prevalence and relief of pain in trauma patients in emergency medical services. Clin J Pain. 2011; 27(7): 587–592. PubMed Abstract \| Publisher Full Text\n\nEbben RHA: Quality of solo ambulance care by physician assistants versus ambulance nurses for non-conveyed patients: An observational study in the Netherlands. DANS. 2021. http://www.doi.org/10.17026/dans-zqw-qbqp" }	[ { "id": "157722", "date": "16 Jan 2023", "name": "Erika Frischknecht Christensen", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThanks for letting me review this paper about care made by physician assistants (PA) vs ambulance nurses in non-conveyance of EMS patients. The topic is of interest in emergency care as new strategies are need and should be investigated. However, physician assistant (PA) is not a well-known category within EMS and the introduction can be improved by explaining this to the reader and how PA’s are expected to improve or change care. Is a solo ambulance like other ambulances for transporting patients or is it simply a car? And if it is an ambulance does the PA alone take the patient onto the trolley in the conveyed cases? Also, the international reader needs information about the Dutch Triage Standard – and how complaints can be converted to ICD-10 diagnoses (table 2)\nThe study aims are ambitious with several outcomes and follow up. All-over, the data material seems sound with EMS patient data sources and questionnaires. The main methodological limitation is the small sample size: the rather low number of patients, especially the low number of PA cases and the low response rate: 1549 solo-ambulances out of 40910 ambulances (2-3%), and only 379 of these solo-ambulances included, with PA in 49 (13%) cases – and follow-up on 165 (44%), and among these only 18 PA. This is contrasting the large number of factors (time of the day, the week, complaints, diagnoses, vital functions (14 factors) analyzed and compared, implying the risk both of type II error due to the low number (as discussed by the authors) and risk of ‘multi-significance’ – finding statistically significant differences just by chance. I recommend focusing on the few most important factors and outcomes. Missing data were treated as random in the analyses but only partly reported in the tables. What are the possible implications of missing data?\nThe follow-up by questionnaires is interesting, and showed that more than half of the patients requested follow-up (and of these 52/90 (58%) within 12 hours) which seems high and may indicate that non-conveyance was not the best decision? Also, half of the respondents reported a lot or very much suffering. There might be a bias due the low response rate, or? Please, discuss.\nIt is reported, both in results, conclusion and abstract that 91.9 % of the follow-up was in the ambulance nurse group – based on which calculations? Seems to correspond to the distribution of 80 ambulance nurses and only 10 PA in the follow-up analyses. Please be very cautious, both in reporting results and in the discussion and conclusions based on the follow-up data.\nIt is appreciated that the patients were asked about their experience, but I did not get access to the appendix.\nFinally, considering the concept of non-conveyance, this study seems to indicate that benefit is almost exclusively for EMS, as only 8% needed an ambulance, while two thirds had follow-up in the ED and almost one third in out-of-hours primary care. Reflections?\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "267207", "date": "07 May 2024", "name": "Hassan Farhat", "expertise": [ "Reviewer Expertise Pre-hospital Emergency Medicine", "Emergency Medicine", "Disaster Medicine", "Epidemiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for this nice piece of work. Although the subject is important, I believe more improvement is needed to make this manuscript approvable. Please find below my improvement suggestion.\nAbstract\nPlease rewrite the abstract background to explain the actual status and challenges rather than starting directly by stating your aim. The method does not state the study period or the type of the study, what you wanted to measure, or which variables. You mentioned, “For initial complaints and the on-scene diagnoses, there were significant differences between the physician assistants and ambulance nurses.” How did you measure that? This should be at least stated in the methodology. Also, there should be a comma after “diagnoses”. The results and the methods of the abstracts need much editing. Please mention the test you used or what are the variables you are measuring. Also, the p-value alone is not informative; I suggest using further metrics such as the CI. “Besides small statistical but not clinically significant differences, this study indicated comparable solo ambulance care provided by a physician assistant or an ambulance nurse concerning the care process, follow-up care and patient experience.” This was unclear. Please edit the conclusion to improve the flow of the language. Overall, if you see the title indicating that you are measuring the quality of the service provided, The quality metrics used to measure it should be clearly defined in the introduction.\n\nIntroduction\nPlease edit the following text to improve the flow for the reader: “Emergency medical services (EMS) face a challenging environment with an increasing number of ambulance deployments1,2. Reasons for this increase are a growing population with more complex healthcare problems and comorbidities, repeated requests for ambulance care, and the request of ambulance care for primary care problems 3–6. Within this increasing demand, there is also a growing population that receives ambulance care without conveyance to another healthcare facility.” When you mentioned, “The non-conveyance decision can be initiated by the ambulance professional (sometimes after consultation with a general practitioner or medical specialist) or the patient and/or their relatives”, are you referring to the worldwide EMS system? Because I would disagree as there are a lot of research articles and reviews mentioning there are various processes. If you are speaking about local context, I would suggest starting from the most general (Worldwide) and then moving to the more specific by explicitly saying which system you are referring to. Please explain why “For general patient populations, the non-conveyance proportion ranges from 3.7–93.7%9.”\n\nMethods\nI suggest that the authors add a process map explaining the workflow of their EMS system functioning after receiving an emergency call, starting from a call until the patient is endorsed. This will help explain the operational plan of the included EMS system. Could you please provide more details about the additional education programs? Does it allow the nurses to provide medicines to the patient? What kind of medicines? Please provide about the non-conveyance modality in your EMS. Is it by the patient only? Or can it be undertaken by the patients and clinicians? Is there any follow-up system? The data analysis needs to be explained in more detail: Please explain which variables you assessed. Which test did you use for each? What were the hypotheses you wanted to test? And did you assess the statistical conditions required for those tests?? Did you account for confounding factors in your analysis?\n\nResults:\nCan you please be consistent in using one or two decimal digits? Please provide the CI with that p-value and explain what hypothesis was confirmed or rejected. Still, the results do not provide details on how you assessed the Quality of your EMS system. All the results provided are descriptive. The title methods and results sections should be revised. Tables 2 and 3 explain the variables; they should be explained earlier, and why did you choose them? And benchmarks? Please explain how you assessed the patient’s experience and its importance in evaluating the quality of a system.\n\nDiscussion\nIt was unnecessary to state the objective again; I suggest removing it and starting your discussion with a more general recapitulation of the implication of the results considering your objectives. I would also suggest discussing the implications of the results considering the socio-demographic characteristics of the population besides the comparison with other studies. Also, since you are assessing the quality, I would suggest using one of the quality control tools in your results and discussing it in the discussion section. I would suggest providing the impact of the study in ensuring more patient-centred care in your system.\n\nThe conclusion has to be re-edited. The flow of the information is not clear. Some of the references were between 1995 and 2011. I suggest you use more recent references as the EMS system worldwide has improved significantly.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/10-357
https://f1000research.com/articles/10-354/v1	07 May 21	{ "type": "Research Article", "title": "Horizontal transfer and evolution of wall teichoic acid gene cassettes in Bacillus subtilis", "authors": [ "Granger Sutton", "Gary B. Fogel", "Bradley Abramson", "Lauren Brinkac", "Todd Michael", "Enoch S. Liu", "Sterling Thomas", "Gary B. Fogel", "Bradley Abramson", "Lauren Brinkac", "Todd Michael", "Enoch S. Liu", "Sterling Thomas" ], "abstract": "Background: Wall teichoic acid (WTA) genes are essential for production of cell walls in gram-positive bacteria and necessary for survival and variability in the cassette has led to recent antibiotic resistance acquisition in pathogenic bacteria. Methods: Using a pan-genome approach, we examined the evolutionary history of WTA genes in Bacillus subtilis ssp. subtilis. Results: Our analysis reveals an interesting pattern of evolution from the type-strain WTA gene cassette possibly resulting from horizontal acquisition from organisms with similar gene sequences. The WTA cassettes have a high level of variation which may be due to one or more independent horizontal transfer events during the evolution of Bacillus subtilis ssp. subtilis. This swapping of entire WTA cassettes and smaller regions within the WTA cassettes is an unusual feature in the evolution of the Bacillus subtilis genome and highlights the importance of horizontal transfer of gene cassettes through homologous recombination within B. subtilis or other bacterial species. Conclusions: Reduced sequence conservation of these WTA cassettes may indicate a modified function like the previously documented WTA ribitol/glycerol variation. An improved understanding of high-frequency recombination of gene cassettes has ramifications for synthetic biology and the use of B. subtilis in industry.", "keywords": [ "wall teichoic acids", "pan-genome", "pan-genome graph", "core genes", "Bacillus subtilis" ], "content": "Introduction\n\nIn a recent paper, we studied the relationship between essential and core genes in Bacillus subtilis ssp. subtilis through a pan-genomic approach. Core genes are the set of genes present in all or almost all strains of a species/subspecies in a pan-genome. Pan-genomes are determined computationally by finding orthologous gene clusters (OGC) between strains based on homology and genome context.1–3 An OGC is the set of genes, with at most one per strain, that have been computationally determined to be orthologs. An OGC is “core” if the number of genes in the OGC exceeds or equals some threshold such as 95% or 100% of the strains in the pan-genome. A node within a pan-genome graph (PGG) is an OGC and if two nodes (OGCs - specifically the genes in the OGCs) are adjacent in the genome of one or more of the pan-genome strains this is considered an edge. While core genes are determined computationally, “essential genes” are experimentally determined genes that render an organism as nonviable if removed in laboratory growth conditions. Such genes are determined experimentally through knockout studies via methods such as random transposon insertion.\n\nFor example, Koo et al.4 and Kobayashi et al.5 computationally and experimentally determined the essential gene set in the gram-positive bacterium Bacillus subtilis ssp. subtilis. For B. subtilis ssp. subtilis, both Koo et al.4 and Kobayashi et al.5 used similar single knockout methods to determine “essential” genes when grown in LB at 37°C. Koo identified 257 essential genes while Kobayashi identified 271 essential genes. The union of these two sets resulted in 305 essential genes.6 Sutton et al.6 mapped these 305 genes to the PGG OGCs using the type strain 168 RefSeq genome NC_000964.3 (BioSample SAMEA3138188) used by Koo et al.4 and Kobayashi et al.5 and 289 were determined to be core OGCs.6 For those genes that were determined to be essential in laboratory conditions but determined not to be core for the subspecies pan-genome, Sutton et al.6 found that some were not truly essential but rather “conditionally essential” due to the presence of other genes such as toxin/antitoxin cognate pairs where the antitoxin is essential in the presence of the toxin gene.\n\nCuriously, eight of the 305 essential genes that were not core genes are involved in the biosynthesis of wall teichoic acid (WTA). These include tuaB (OGC 4729 present in 85 of 108 genomes), mnaA/yvyH (OGC 4735 present in 84 of 108 genomes), tagH (OGC 4744 present in 84 of 108 genomes), tagG (OGC 4745 present in 35 of 108 genomes), tagF (OGC 4746 present in 35 of 108 genomes), tagD (OGC 4748 present in 35 of 108 genomes), tagA (OGC 4749 present in 35 of 108 genomes) and tagB (OGC 4750 present in 35 of 108 genomes). By examining the PGG we determined that genes homologous but diverged from the type strain 168 WTA genes were present at the same relative genome location but were contained in other OGCs. In order to understand the evolution of these essential WTA genes that are not contained in single OGCs, more diverged protein orthology was used to overcome the strong OGC constraint of a minimum of 90% identity over 90% of gene length at the nucleotide level. Our analysis indicates that the WTA genes have a high level of variation possibly due to horizontal gene transfer via recombination as entire cassettes and smaller regions in B. subtilis ssp. subtilis.\n\nWTA genes are involved in production of anionic glycopolymers required for consistent cell shape and division.7,8 Mutants deficient in WTA biosynthesis show increased sensitivity to temperature and certain buffer components and cells tend to aggregate in culture.9–14 While the WTA genes have been shown to be dispensable,11 the resulting cells have abnormal morphology and show impaired growth and reproduction. B. subtilis and S. aureus mutants deficient in lipoteichoic acid (LTA) biosynthesis can be obtained but only if grown under a narrow range of conditions; they are temperature sensitive and exhibit severe growth defects.9,10\n\nTaken together, LTAs and WTAs create what has been aptly described by Neuhaus and Baddiley as a “continuum of negative charge” that extends from the bacterial cell membrane beyond the outermost layers of peptidoglycan.15 New pathogenesis-related functions for WTAs have also been realized and it has been suggested that the biosynthetic enzymes that make these polymers are targets for novel antibacterial agents.16,17 Indeed, the first WTA-active antibiotic18 acts by blocking the export of WTA to the extracellular surface. The chemical structures of WTAs vary in gram-positive bacteria but the most common structures are glycerol or ribitol phosphate repeats.19–26 B. subtilis can make poly (glycerol phosphate) or poly (ribitol phosphate) WTAs depending on the strain,27 while S. aureus strains primarily make poly (ribitol phosphate) WTAs.28–31\n\nThe ribitol WTA genes (tar) were thought to distinguish B. subtilis ssp. spizizenii strains from the glycerol WTA genes (tag) contained in B. subtilis ssp. subtilis strains based on functional studies in B. subtilis strain W23 which is a B. subtilis ssp. spizizenii strain and B. subtilis strain 168 which is a B. subtilis ssp. subtilis strain.32 Sequencing of the W23 tar genes revealed eight open reading frames in two adjacent divergently transcribed operons, tarABIJKL and tarDF, where tarA, tarB, tarD and tarF have clear homology to their counterparts tagA, tagB, tagD and tagF while tarI, tarJ, tarK and tarL have no obvious homology to tag genes. The four conserved tar/tag genes appear to construct the basic core of the teichoic acid structure although for tar/tagF the function is somewhat different since tagF is much longer than and shares only C-terminal homology with tarF.33 The four tar genes which are not conserved presumably are specific to the ribitol modifications to teichoic acids. The tag genes are also organized in two adjacent divergently transcribed operons tagABC and tagDEF where tagC and tagE have no obvious homology to tar genes.32 More recently it was determined that the ribitol/glycerol distinction of tar versus tag genes is not distinguishing between spizizenii and subtilis subspecies but rather either subspecies can contain one or the other.34\n\n\nMethods\n\nFor the B. subtilis ssp. subtilis pan-genome we selected strains with complete genomes in RefSeq.35 We restricted our analysis to complete genomes to ensure that missing genes due to incomplete genome sequencing/assembly did not affect the approach or results. We limited our choice to RefSeq for two reasons: RefSeq performs a series of quality checks to remove dubious genome assemblies, and the initial pan-genome construction depends upon reasonably consistent annotation which RefSeq provides. We extracted the genomes based on organism name: Bacillus subtilis (we did not specify subspecies, since for many RefSeq genomes a subspecies is not given). For each pan-genome we then compared the genomes using a fast Average Nucleotide Identity (ANI) estimate generated using MASH.36 We used type strains and ANI to determine which of these genomes were the desired organism. We also used ANI to remove very closely related strains to reduce oversampling bias (for example, for the B. subtilis type strain, 168, has at least eight genomes in RefSeq). We used GGRaSP37 to choose a single medoid sequence from any complete linkage ANI cluster with a threshold of 0.01% or 1/10,000 base pair difference. The strain 168 medoid genome is the Entrez reference genome for the B. subtilis type strain (GenBank sequence AL009126.3, BioSample SAMEA3138188, Assembly ASM904v1/GCA_000009045.1) which can be used to map the Koo et al.4 and Kobayashi et al.5 results.\n\nUsing this approach, for B. subtilis 143 genomes were downloaded from RefSeq. Of these 132 genomes were determined to be B. subtilis ssp. subtilis based on type strains and ANI. The minimum ANI between any pair of the 132 B. subtilis ssp. subtilis genomes was 97.28% whereas the maximum ANI of any of the 11 other genomes to the 132 genomes was 95.73% providing good separation between the other subspecies. The 132 genomes were reduced to 109 genomes after removing redundant strains. Finally, we removed strain delta6 (BioSample SAMN05150066) because it is known to have been engineered to remove multiple genes. Thus, we were left with 108 B. subtilis genomes (Supplementary Table 1).\n\nFor B. subtilis ssp. subtilis the initial pan-genome was based on the RefSeq annotation of these genomes. The pan-genome was generated using the pan-genome pipeline at the J. Craig Venter Institute (JCVI) at the nucleotide level using default parameters with the exception that a minimum of 90% identity and 90% length for pairwise Blast matches were used to prevent possible clustering of non-orthologous genes.38 This produced ortholog gene clusters (OGC) using gene context1 as well as a Pan-Genome Graph (PGG).2 The PGG has two main components: nodes representing OGCs, and edges representing the sequence between genes and the order and orientation of the genes in the genomes. We updated the code repository for the JCVI pan-genome pipeline with a script: iterate_pgg_graph.pl which calls pgg_annotate.pl for the genomes in the existing PGG in order to ensure consistent annotation of the genomes and iterates until the PGG stabilizes. The script pgg_annotate.pl uses an existing PGG to assign regions of a genome to nodes of the graph. This is done by blasting the medoid gene sequence for the OGC the node represents against the genome and then uses Needleman-Wunsch39 to extend the alignment if needed. If there are conflicting blast matches, then the matches are resolved based on which matches are consistent with the structure of the PGG which encapsulates gene context across the entire pan-genome. Once the nodes of the PGG are mapped to each of the genomes in the pan-genome a new version of the PGG is intrinsic and then explicitly extracted. This process is iterated to stability. This ensures that each genome is consistently annotated so that missing genes are not due to inconsistent annotation.\n\nThe medoid nucleotide sequence for each of the 144 OGCs found in any of the WTA cassettes was translated into peptide sequences. These 144 peptide sequences were combined into a multifasta file to create a peptide Blast database (makeblastdb -in WTA_prot.fasta -dbtype prot). A peptide level all versus all Blast search40 of these 144 peptide sequences was performed (blastp -query WTA_prot.fasta -db WTA_prot.fasta -out tmp -task blastp -evalue 0.000001 -outfmt \"6 qseqid sseqid pident qstart qend qlen sstart send slen evalue bitscore stitle\"). Potential protein ortholog matches were retained if the percent identity was ≥40% and the length of the match was ≥80% of the shorter protein. Matches with smaller bit scores than the first match to a protein from the same clade (i.e. a paralog) were not retained. More limited matches were retained for tagF matches in an attempt to determine possible orthologs but they were not treated as likely orthologs. The protein ortholog Blast matches are in Supplementary Table 7 and the probable protein orthologs are in Table 1.\n\nColumn 1 is the gene name/symbol. Column 2 is the set of OGCs determined to be orthologs at the protein level. Column 3 is the number of the 108 strains in the PGG which contain one of the protein level orthologs. Column 4 is OGC medoid sequence RefSeq annotation for one of the protein level orthologs.\n\nThe OGC subpatterns show some limited recombination within the WTA cassettes but most recombination seems limited to the entire cassette. Column 1 is the region between core OGCs within the WTA cassette. Column 2 is an OGC subpattern. Columns 3-9 indicate the number of strains within a clade that has the given OGC subpattern for that row. The rows are ordered relative to their order in the WTA cassette from core OGC 3712 to core OGC 3756.\n\nThe extracted nucleotide WTA cassette sequences were placed in a multifasta file. The sequences were aligned using Mafft41 (mafft –reorder WTA_cassette.fasta > WTA_cassette.mafft). The alignment was trimmed of gappy columns using trimal42 (trimal -in WTA_cassette.mafft -out WTA_cassette.trim -gt 1 -fasta). The trimmed alignment was used as input to RaxML43 run from TOPALi v2.544 with default parameters to generate a phylogenetic tree.\n\nPhylogenetic trees were generated from the pan-genome using complete linkage hierarchical clustering of pairwise Jaccard distance and genome ANI distances. The resulting trees were rendered using the Interactive Tree of Life (iTOL).45–47 A linear illustration of the WTA cassettes was generated using the SimpleSynteny tool.48\n\n\nResults\n\nFor our 108 strain pan-genome (Supplementary Table 1), the WTA genes in all genomes are colocalized and bounded by two core genes: yvyE (OGC 3712) and ribZB (OGC 3756). For all but one genome, this region is contiguous and therefore can be considered as a cassette. For strain N1-1 (SAMN10225190), the cassette is split into two nearby contiguous pieces due to an inversion between two inverted copies of an IS1182 transposon, one inside the cassette and one outside the cassette. This could be a real rearrangement or an assembly error. For the purpose of our analysis we treated these two pieces as a single cassette as if the inversion had not taken place.\n\nWe extracted the PGG annotation, which assigns genome coordinates to OGCs, for just the WTA cassettes from the complete PGG annotation of the 108 genomes (Supplementary Table 2). There were a total of 144 OGCs which were present in one or more of the WTA cassettes which we extracted from the overall set of OGCs (Supplementary Table 3). We also extracted the set of edges between these OGCs from the PGG (Supplementary Table 4). Based on the presence or absence of each of the 144 OGCs in a strain we calculated the pairwise Jaccard distance as a measure of WTA gene content dissimilarity (Supplementary Table 5). The strains were then classified into seven clades (I-VII) based on the Jaccard distance (Figure 1). Clades I-VII have 43, 2, 3, 1, 23, 35, and 1 strain(s), respectively. To determine whether the gene content difference was due to evolutionary inheritance, we determined the pairwise average nucleotide identity (ANI) similarity between strains (Supplementary Table 6) which is considered to be strongly correlated with evolutionary distance (Figure 2). Since the WTA gene-content based clades do not form the same clades under ANI distance, but rather intermix, something beyond straight inheritance is accounting for the WTA gene content within strains.\n\nWe distinguished seven clades. Clade I (blue) is the ribitol WTA consistent with that found in strain W23. Clade II (purple) and clade III (orange) appear to also be ribitol WTA based on the presence of tarI (OGC 5434), tarJ (OGC 5435), tarK (OGC 5436) and tarL (OGC 5437). Clade IV (yellow) may be a ribitol WTA based on a shorter tagF gene. Clade V (red) may be a glycerol WTA based on a longer tagF gene. Clade VI is the type strain glycerol WTA. Clade VII (teal) is missing many key WTA genes (tag/tarBADFG) so it is unclear how the one strain in that clade is constructing WTAs. The 10 medoid strains used in Figure 3 are in bold.\n\nsubtilis in the pan-genome based on complete linkage hierarchical clustering of pairwise genome ANI distances (100 – ANI). The colors indicate which of the seven WTA gene content clades a strain is in from Figure 1. The 10 medoid strains used in Figure 3 are in bold.\n\nTo determine orthology beyond the strict orthology of the OGCs, we used protein level Blast (blastp) to compare the medoid gene sequence of each of the 144 WTA OGCs against the other 143 WTA OGC medoid gene sequences (Supplementary Table 7). The medoid gene sequence of an OGC is the gene sequence with the lowest summed pairwise homology distances as determined by Blast to all other gene sequences in the OGC. The ribitol specific genes tarI (OGC 5434), tarJ (OGC 5435), tarK (OGC 5436) and tarL (OGC 5437) had no obvious OGC protein orthologs. Likewise, the glycerol specific genes tarC (OGC 4751) and tarE (OGC 4747) had no obvious OGC protein orthologs. Based on the ribitol and glycerol specific OGCs, three clades (I, II and III) appear to be ribitol WTAs, one clade (VI) appears to be glycerol WTA, and three clades are an indeterminate WTA type.\n\nWithin the WTA cassette there are three adjacent OGC pairs (for a total of six OGCs) which are conserved across all 108 strains: tagO-tuaH (OGCs 3721-3722), lytD-pmiA (OGCs 3749-3750) and gerBC-ywtG (OGCs 3753-3754), in that order from bounding OGC 3712 to bounding OGC 3756. Each of these highly similar OGC pairs contain similar DNA sequences which allow for homologous recombination events to occur between strains from different clades. We did not explicitly examine possible recombination between strains of the same clade. We illustrate the basic OGC structure within the WTA cassette for the seven clades shown in Figure 1 in the linear alignment shown in Figure 3 of the WTA cassettes from the 10 medoid genomes determined by ANI and shown in bold in Figure 2.\n\nArrows indicate individual WTA genes drawn to scale with order and orientation maintained. The coordinates for the WTA cassettes in SAMN08707592 and SAMN08707595 which are located on the opposite strand, were reversed for rendering. Genes between strains belonging to the same OGC are joined vertically by correponding colored lines.\n\nWe examined OGC and OGC protein ortholog based evidence of recombination between clades for each clade. We did this by determining the OGC patterns for each of the 108 strains across the WTA cassette (Supplementary Table 8). An OGC pattern is simply the order of OGCs across the WTA cassette for a given strain from OGC 3712 to OGC 3756. For identical OGC patterns we collapsed identical columns for a simpler presentation in Supplementary Table 9. When analyzing the OGC patterns, we specifically looked for common subpatterns, a subinterval of the OGC pattern, to determine possible recombination events. An OGC pattern or subpattern is shown as a parentheses bounded comma separated list of OGCs where OGCs with consecutive numbers are indicated with a hyphen (e.g. 4724-4730,5418-5421,4734-4735,5422-5424,4736,4744, 5425). A null subpattern indicated by () means there are no OGCs for that subpattern. We looked specifically at the four subregions of the WTA between the bounding OGCs and the three conserved OGC pairs. We designated these four regions based on their bounding OGCs: 3712-3721, 3722-3749, 3750-3753, and 3754-3756.\n\nFor the 23 strains in clade V, there are eight OGC patterns (Supplementary Table 9, Supplementary Table 8, Table 2). For 22 strains forming seven OGC patterns all non-core OGCs are specific to clade V with only minor OGC variation in all regions except 3750-3753. For these 22 strains no recombination appears to occur within regions 3712-3721, 3722-3749, and 3754-3756. Within the 3750-3753 region recombination does appear to be occurring with four OGC patterns and 13 strains with OGC subpattern (3751-3752); whereas four OGC patterns and 10 strains have OGC subpattern (4752-4753). In region 3750-3753, there are only three OGC subpatterns for all 108 strains: (3751-3752), (4752-4753), and (5718,3752). Pattern (3751-3752) is in 13/23 clade V strains and 12/85 of other clade strains; whereas, pattern (4742-4753) is in 10/23 clade V strains and 72/85 of other clade strains. This might indicate that OGC subpattern (3751-3752) is ancestral (before recombination) for clade V and OGC subpattern (4752-4753) is ancestral for the other clades. In the 3754-3756 region, there is only one OGC subpattern (3755). Clade V strain PJ-7 (SAMN10225146) is the only clade V strain with a different OGC subpattern in region 3712-3721 having instead the same OGC subpattern as the other clades. Strain PJ-7 is an outlier in the ANI tree compared to the rest of clade V. Region 3712-3721 appears to be ancestral for PJ-7, but the rest of its WTA cassette appears to have been acquired via homologous recombination with a clade V strain or from the same source the ancestral clade V strain acquired its divergent WTA cassette. All clade V strains except PJ-7 have OGC subpattern (3713-3718,9158,3719-3720) in region 3712-3721, whereas strain PJ-7 and all the other clades have OGC subpattern (4723) in region 3712-3721. OGC 3713 has protein orthology to OGC 4723 (Table 1).\n\nFor all clades except clade V, region 3712-3721 has OGC subpattern (4723) and region 3722-3749 begin with OGC subpattern (4724-4730). For the 43 strains in clade I, there are seven OGC patterns in the WTA cassette (Supplementary Table 9, Supplementary Table 8, Table 2). In region 3722-3749, all seven OGC subpatterns begin (4724-4730,5418-5421,4734-4735,5422-5424,4736,4744,5425) but then diverge near the tarF gene. There are four OGC subpatterns containing the tarF gene: (4738-4741), (5994-5998), (5426-5430), and (7395-7397). For the most part these four OGC subpatterns are unique to clade I and do not appear to be due to recombination. OGC subpatterns (4738-4741) and (5994-5996) are shared by some clade VI strains but in a non-orthologous location and as we discuss below OGCs 4740-4741 appear to be a mis-annotation for clade I strains where a tarF gene should be annotated instead. By a non-orthologous location, we mean that the OGCs are not occurring in the same OGC/gene context for the different clades possibly indicating that they are not truly orthologous. After the tarF gene, the remainder of region 3722-3749 has the same OGC subpattern (5431-5437) for all clade I strains. As discussed above, there does appear to be recombination in the 3750-3753 region for clade I strains. In the 3754-3756 region, there are two OGC subpatterns: (4754) and (5438). OGC subpattern (5438) occurs in only 5/43 clade I strains, 1/2 clade II strains, and in no other clades. With the limited number of clade II strains it is impossible to guess if OGC subpattern (5438) is ancestral for clade II and recombining with clade I. Alternatively, clade I and clade II may have both acquired OGC subpattern (5438) independently from an unknown source.\n\nFor the 35 strains in clade VI, there are seven OGC patterns (Supplementary Table 9, Supplementary Table 8, Table 2). In region 3722-3749, all seven OGC subpatterns begin (4724-4736) but then diverge after the gtaB gene but before the tagH gene. There are four OGC subpatterns: (10139,4737-4743), (10139,4737,5994-5996,7091-7093,4743), (10139,4737,5994-5996,7091-7093,4743), and () containing no OGCs. For the most part these four OGC subpatterns are unique to clade VI and do not appear to be due to recombination. OGC subpatterns (4738-4741) and (5994-5996) are shared by some clade I strains but in a non-orthologous location as discussed above. The remainder of region 3722-3749 has the same OGC subpattern (4744-4751) for all clade VI strains. As discussed above, there does appear to be recombination in the 3750-3753 region for clade VI strains. In the 3754-3756 region, there is only OGC subpattern (4754).\n\nFor clades II, III, IV, and VII recombination based on OGC patterns is harder to evaluate since they only have 2, 3, 1, and 1 strain(s), respectively. For clade VII, the OGCs which are not unique to clade VII are common to most other clades. For clade IV, this is also true with the exception of OGC subpattern (7625-7631) which is shared with clade III but it is unclear if this is due to shared ancestry or recombination. For clade III, OGC patterns are mixed between that seen for clades I and VI with some unique OGCs mixed in but there is no obvious recombination pattern except in region 3754-3756 where there are two OGC subpatterns: (3755) and (4754). For clade II, the OGC patterns are very similar to clade I with some unique OGCs and no evidence of recombination except in region 3754-3756 where there are two OGC subpatterns: (5438) and (4754).\n\nFor the eight essential WTA genes that did not have core OGCs, we combined the protein ortholog OGCs (Table 1) showing: tuaB, mnaA/yvyH, tagG, and tagH are in all 108 strains; tagD, tagA, and tagB are in 107 strains (missing from the one clade VII strain); and tagF is in only 87 strains. We investigated why tagF is missing from 20 strains besides the clade VII strain also missing tagDAB. It is also missing from all clade II and III strains and from 15 clade I strains. For the 15 clade I strains two short proteins (OGCs 4740-4741) have homology to tagF in nonoverlapping regions but many clade VI strains have both OGCs 4740-4741 and 4746 (tagF). We searched the entire genomes of the strains missing an obvious tagF ortholog using tblastn to search at the peptide level the tag/tarF medoid proteins for OGCs 4746 (tagF), 5988 (tarF), and 5430 (tarF). For the 15 clade I genomes both 5998 and 5430 matched full length at 65% and 70% identity to a region adjacent to OGC 5431 and overlapping the OGCs 4740 and 4741. This indicates that these 15 genomes are misannotated in this region. Unfortunately, the PGG-based annotation pipeline enforces consistency of annotation but not correctness of annotation. The original RefSeq annotation had OGCs 4740 and 4741 for 24 and 23 strains respectively, while the tagF ortholog for the same region was annotated for 15 strains. The PGG refinement process will choose between competing OGC annotation for a region based on predominance which resulted in OGCs 4740 and 4741 replacing the tagF ortholog annotation for those 15 strains. This results in 39 genomes being annotated with OGCs 4740 and 4741: 15 from clade I which do not have an alternative tagF ortholog and 24 from clade VI which do have an alternative tagF ortholog (OGC 4746). In these 24 clade VI strains the entire tagF ortholog (1185 bp) from the 15 clade I strains is not present but only the C terminal region (bps 654-1185) which contains OGCs 4740 and 4741 matches with 93% sequence identity. One possibility is that this region was transferred during homologous recombination but due to the presence of another tagF gene in the genome this particular tagF devolved in these 24 strains leaving two small open reading frames annotated as hypothetical proteins. For the clades II and III genomes no obvious tagF ortholog was found using tblastn but significant hits to OGC 5437 (tarL) had low homology and significant but lower homology was seen for several other WTA cassette OGCs. It is not clear whether one or more of these homologs may be providing the needed tagF functionality. For the one clade VII strain the entire core WTA machinery is missing (tag/tarBADF). Based on the literature one would expect this to be an engineered strain with limited viability and abnormal cell morphology but it is a soil isolate with normal cell morphology.49 It is an interesting question whether the one clade VII strain is deficient in WTAs but compensating with LTAs.\n\nTo better understand the homologous recombination occurring in the WTA cassettes, we extracted the nucleotide sequences for all 108 cassettes, aligned them, trimmed them to remove columns predominated by gaps, and generated a maximum likelihood tree (Figure 4). Since clade V strains cluster on one branch of the tree with a much higher bootstrap value than any other branch, they clearly have a very diverged cassette from other strains. The intermixing of other clades in the remainder of the tree possibly indicates active homologous recombination within the WTA cassettes of those clades. The clade V WTA cassette in contrast appears to have been acquired in whole from some more distant organism and is not actively recombining within the WTA cassette with any other strains in other clades. The one exception to this is for strain PJ-7 (SAMN10225146) which does not have region 3712-3721 of the clade V WTA cassette. It is an open question whether this one outlier clade V strain acquired its WTA cassette from some other clade V strain or independently from the same organism as the other clade V strains.\n\nMidpoint rooted and branchlengths ignored. Strains are color coded by WTA clade membership (I-VII). Numbers at nodes represent bootstrap support. The 10 medoid strains used in Figure 3 are in bold.\n\n\nDiscussion\n\nA pangenome analysis of B. subtilis ssp subtilis indicates that there are seven diverged cassettes of WTA genes (clades I-VII) in B. subtilis ssp. subtilis. Of these only the type strain glycerol WTA gene cassette, clade VI, is well characterized in the literature, alongside significant work on the ribitol WTA gene cassette of clade I. Based on the ribitol and glycerol specific OGCs three clades (I, II and III) appear to be ribitol WTAs, one clade (VI) appears to be glycerol WTA, and three clades are an indeterminate WTA type. Clade V may be a glycerol WTA based on a longer tagF gene which is associated with glycerol WTAs versus a shorter tagF for ribitol WTAs. Clade IV may be a ribitol WTA by the same reasoning with a shorter tagF gene. Clade VII is missing many key WTA genes (tag/tarBADFG) so it is unclear how the one strain in that clade is constructing WTAs or if it is compensating with LTAs. Additional biochemical characterization of the cell walls for these clades would be required.\n\nMore recently it was determined that the ribitol/glycerol distinction of tar versus tag genes is not distinguishing between spizizenii and subtilis subspecies but rather either subspecies can contain one or the other.34 Having aligned tar/tagBAD genes from multiple B. subtilis ssp. subtilis strains, Ahn et al.34 determined that there were two main clades, one for ribitol WTA (group A) and one for glycerol WTA (group B), with three outliers not in either clade. Group A included four of our clade I strains (BAB-1, UD1022, OH 131.1, BSP1). Group B included four of our clade VI strains (RO-NN-1, BSn5, 168, SG6). One outlier (strain VV2), is one of our clade II strains. Another outlier (strain BEST195), is one of our clade V strains.\n\nThe WTA gene cassette is highly variable in B. subtilis ssp. subtilis, much more so than other essential or core genes.6 This does not appear to be an ancient evolutionary split of strain subtypes, although inheritance is an obvious component, but rather the result of homologous recombination of WTAs as an entire cassette or just regions within the cassette. This method of rapid evolution is often in response to environmental pressure (e.g. phage infection). The WTAs exposed on the cell surface have been shown to be potential phage targets and variation in the WTAs can determine phage susceptibility50–52 and therefore strain fitness. Likewise, antibiotic susceptibility has been shown to be a driving factor in strain fitness in the wild and WTA biosynthetic proteins are the targets of some antibiotics and further study of these novel WTA OGCs may lead to novel antibiotics.53–57 WTAs can also modulate specificity of species and strains that can effectively engage in horizontal gene transfer and thereby acquire antibiotic resistance or virulence traits.58 WTAs are also involved in host immune system evasion.59–62\n\nThe high rate of recombination and genetic tractability in B. subtilis make it a model organism for biological engineering. Discovery of novel core OGCs using a pan-genome approach can help identify genomic regions capable of excluding gene insertions or non-essential OGCs ready for deletion. The high degree of essential gene conservation in the WTA cassette suggests that they might not be easily deleted. The high level of variation in the WTA cassette is intriguing and a possible target for engineering since it appears to be under adaptive pressure. By naturally rearranging the WTA cassette B. subtilis may be able to occupy new niches where acquisition of WTA genes not susceptible to certain antibiotics or phages is advantageous. Conversely, biological engineers might be able to recombine genes conferring antibiotic susceptibility to expand the number of usable antibiotic genes required for manipulating multiple endogenous loci concurrently.\n\n\nConclusion\n\nReduced sequence conservation of the WTA cassettes in the seven clades we determined may indicate a modified function like the previously documented WTA ribitol/glycerol variation found in two of those clades. This WTA variation poses a number of questions about function, response to environmental pressure, and potential engineering targets as discussed above. An improved understanding of high-frequency recombination of WTA gene cassettes has ramifications for synthetic biology and the use of B. subtilis in industry.\n\n\nData availability\n\nFigshare: Underlying data for ‘Horizontal transfer and evolution of wall teichoic acid gene cassettes in Bacillus subtilis’, https://doi.org/10.6084/m9.figshare.14132192.v1\n\nThis project contains the following underlying data:\n\n• Table 1. The protein level orthologous OGCs within the WTA cassettes. Column 1 is the gene name/symbol. Column 2 is the set of OGCs determined to be orthologs at the protein level. Column 3 is the number of the 108 strains in the PGG which contain one of the protein level orthologs. Column 4 is OGC medoid sequence RefSeq annotation for one of the protein level orthologs.\n\n• Table 2. OGC subpatterns for the WTA cassettes across clades I-VII. The OGC subpatterns show some limited recombination within the WTA cassettes but most recombination seems limited to the entire cassette. Column 1 is the region between core OGCs within the WTA cassette. Column 2 is an OGC subpattern. Columns 3-9 indicate the number of strains within a clade that has the given OGC subpattern for that row. The rows are ordered relative to their order in the WTA cassette from core OGC 3712 to core OGC 3756.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC BY 4.0).\n\nFigshare: Underlying data for ‘Horizontal transfer and evolution of wall teichoic acid gene cassettes in Bacillus subtilis’, https://doi.org/10.6084/m9.figshare.14132192.v1\n\nThis project contains the following extended data:\n\n• Supplementary Table 1\n\n• Supplementary Table 2\n\n• Supplementary Table 3\n\n• Supplementary Table 4\n\n• Supplementary Table 5\n\n• Supplementary Table 6\n\n• Supplementary Table 7\n\n• Supplementary Table 8\n\n• Supplementary Table 9\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC BY 4.0).", "appendix": "Acknowledgements\n\nThe authors would like to thank IARPA for sponsoring this research.\n\n\nReferences\n\nFouts DE, Brinkac L, Beck E, et al.: PanOCT: automated clustering of orthologs using conserved gene neighborhood for pan-genomic analysis of bacterial strains and closely related species. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWinstel V, Liang C, Sanchez-Carballo P, et al.: Wall teichoic acid structure governs horizontal gene transfer between major bacterial pathogens. Nat Commun. 2013; 4: 2345. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAtilano ML, Yates J, Glittenberg M, et al.: Wall teichoic acids of Staphylococcus aureus limit recognition by the drosophila peptidoglycan recognition protein-SA to promote pathogenicity. PLoS Pathog. 2011; 7: e1002421. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGautam S, Kim T, Lester E, et al.: Wall teichoic acids prevent antibody binding to epitopes within the cell wall of Staphylococcus aureus. ACS Chem Biol. 2016; 11: 25–30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSukhithasri V, Nisha N, Biswas L, et al.: Innate immune recognition of microbial cell wall components and microbial strategies to evade such recognitions. Microbiol Res. 2013; 168: 396–406. PubMed Abstract \| Publisher Full Text\n\nSutton G: Figshare: Underlying data for ‘Horizontal transfer and evolution of wall teichoic acid gene cassettes in Bacillus subtilis’2021. Publisher Full Text" }	[ { "id": "86085", "date": "18 Jun 2021", "name": "Jeffrey B. Jones", "expertise": [ "Reviewer Expertise plant-microbe interactions", "bacterial pathogenesis" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript is well written and provides ample support for evolution within the WTA gene cassette. Although the authors have provided considerable information on the relationships of the genes in the different clades, I wonder if it would be appropriate to provide an ANI matrix comparing all strains to show relationships between the clades and the ANI values between strains. I suspect although I may be incorrect that these clades may strongly relate to the relative ANI values. It may help in explaining the extent of recombination or perhaps refute that idea. I am more familiar with ANI values for Gram negative bacteria and although values around 97 are relatively close values above 99% show much tighter relationships and may help in explaining the extent of recombination. Although they state the importance of these findings for synthetic biology using this bacterium, it would be helpful if they would provide more information on how.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6824", "date": "22 Jun 2021", "name": "Granger Sutton", "role": "Author Response", "response": "Thank you for the feedback. We're not sure exactly what form of ANI matrix you are requesting. We provide a whole genome all versus all ANI matrix for the 108 genomes as Supplementary Table 6 and Figure 2 shows the tree based on this matrix. Perhaps you want us to highlight this more in the text? We will attempt to be more explicit about applications for synthetic biology in our revision." } ] }, { "id": "101882", "date": "16 Dec 2021", "name": "José Escudero", "expertise": [ "Reviewer Expertise Antibiotic resistance", "horizontal gene transfer", "integrons", "protein evolution", "recombination" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this manuscript the authors report discrepancies between the core genome of B. subtilis and its set of essential genes. Some of the genes that do not match encode the WTA synthesis pathways, which are essential. The reason for the discrepancies, they argue, is that horizontal gene transfer and homologous recombination have led to extensive exchange in these genes leading some alleles not to be recognised as the same OGC and hence not be considered part of the core genome. This type of mosaicism has been described in S. pneumoniae quinolone resistance determining regions (QRDRs) by the group of De la Campa, so I believe maybe mentioning it would be relevant to provide context.\nI find this work to be well carried out and show results that are relevant. I nevertheless find it challenging to follow and believe it would benefit from extensive rewriting to make the language less specialised. Rewriting would especially benefit the results section where a higher context story, rather than a point by point enumeration of results, could be easier to understand, and the details would still be available in the table provided. I would also suggest to include an introductory figure showing, among other things, the basic genetic structure of the WTA genes in a type strain such as Bs168. Maybe including subtilis and spizizenii subspecies to compare these sets of genes would be even better. I would also suggest to change the figure legend titles to sentences telling the result rather than describing the figure and methodology. The description and methodology can go in the figure legend after the title.\nAltogether, I believe that the results are relevant but need to be told in a more clear way to reach a broader audience.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "101224", "date": "11 Jan 2022", "name": "Patrick Eichenberger", "expertise": [ "Reviewer Expertise Genomics and molecular biology of endospore-forming bacteria" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article addresses an interesting question by investigating the evolution of wall teichoic acid (WTA) genes in B. subtilis. It is already known that WTA plays a key role in B. subtilis during growth; however, the molecular composition of theses anionic polymers vary from strain to strain. For instance, the type strain (168) makes polymers of glycerol, whereas another frequently studied strain (W23) makes polymers of ribitol. By determining the gene composition and patterns of conservation within the cassette encoding the different enzymes in the WTA synthesis pathway, the authors shed light on the evolutionary history of B. subtilis strains.\nUnexpectedly, there is no clear phylogenetic distinction between poly-ribitol and polyglycerol producing strains, thus strongly suggesting that horizontal gene transfer and recombination within the WTA locus is frequent. As already mentioned by the original reviewer, ample support is provided in the article for this hypothesis. As suggested in the previous round of review, the authors have now added an Average Nucleotide Identity (ANI) analysis, whose outcome is also consistent with the expectations.\nThe methods are explained in detail and with clarity. My only suggestion would be to rephrase the following statement on p.3 “Curiously, eight of the 305 essential genes that were not core genes”. If I understood correctly, of the 305 essential genes, only 8 are not core genes, but the sentence seems to imply that none of the 305 essential genes were core genes and that the focus of the paper will be on 8 of these genes.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-354
https://f1000research.com/articles/7-1619/v2	12 Oct 18	{ "type": "Research Article", "title": "Multilevel analysis concerning the relationship between social vulnerability and the healthy use of leisure time in children and adolescents in Argentina: A national population-based study", "authors": [ "Daniela Moyano", "Zarina Forclaz", "Raúl M. Chaparro", "Akram Hernández-Vásquez", "Nilda R. Perovic", "Daniela Moyano", "Zarina Forclaz", "Raúl M. Chaparro", "Nilda R. Perovic" ], "abstract": "Background: Leisure time is a human right and has to be considered part of any health promotion initiative aimed at children and adolescents. The objective of this study was to analyze the relationship between social vulnerability and the healthy use of leisure time in children and adolescents in urban contexts of Argentina, in 2012. Methods: A cross-sectional and analytical study using data from the Module on Activities of Girls, Boys and Adolescents of the Annual Urban Household Survey was carried out. In this survey, a self-administered instrument was applied to 25,915 individuals aged from 5 to 17. A Social Vulnerability Index (SVI) was developed. Association was estimated by multilevel logistic regression. Results: Children and adolescents use most of their leisure time to carry out school activities (90.1%) with art activities having the lowest percentage (21.8%). In the multilevel models on the relationship between a high SVI and non-performance of socialization activities, the OR was 1.99 (p = 0.002, 95% CI: 1.28-3.12). The association between high SVI and non-use of ICT gave an OR of 14.17 (p < 0.001, 95% CI: 5.13-39.17), and between high SVI and non-use of internet, an OR of 21.89 (p < 0.001, 95% CI: 7.50-63.88). Conclusions: A high SVI negatively impacts on some healthy activities of leisure time for children and adolescents in Argentina. The SVI could be a useful tool to guide health promotion initiatives in this population.", "keywords": [ "Children", "Adolescent", "Recreation", "Social Vulnerability", "Health Promotion", "Argentina" ], "content": "Introduction\n\nLeisure time allows children and adolescents to participate in a diverse range of activities that contribute to developing their identity, improve their self-regulation and express their interests. Several studies show that extracurricular activities benefit the positive development of the child-adolescent population1–3.\n\nIn recent decades, Argentina has made notable progress in expanding the rights of children and adolescents through the adoption and promulgation of various laws, and the adoption, and ratification of the Rights of the Child. Despite the progress made in the legal and institutional framework in 2011–2012, it was observed that 30.1% of children in Argentina were affected by multidimensional poverty4.\n\nSocial vulnerability in children and adolescents is a central element in the definition of social protection policies5 that seek to improve the quality of life in children and adolescents6 and promote healthy free-time uses7.\n\nToday, leisure time is considered a right. This is claimed by the Convention on the Rights of the Child8, in which it is understood as a time for rest and leisure7, and it is considered a necessary element in the approach to health promotion9; but it is also one of the main social determinants of health10.\n\nPrevious studies indicate that the use of leisure time is related to social vulnerability in children and adolescents11–13. In these, it is observed that as the social stratum diminishes, the low performance of extracurricular recreational activities (sports, art and cultural) increases and the probability of not having access to this type of incentives increases14. The social inequality gap in socialization opportunities is significant and clearly regressive for children and adolescents living in poverty in Argentina14. On the other hand, gender socialization may be an important mechanism to both understand and counteract observed differences in participation in free-time activities among genders12,14,15.\n\nThe scarce evidence documented in the field explored in the Argentina and Latin America highlights the importance of the production of knowledge of the impact of social vulnerability on the healthy use of leisure time in children and adolescents in order to contribute to the quality of life and health of this group from a holistic and integral perspective.\n\nThe main objective of this study was to analyze the relationship between social vulnerability and the healthy use of leisure time of children and adolescents in urban contexts in Argentina in 2012.\n\n\nMethods\n\nThis was a cross-sectional, analytical study of a secondary database of the Module on Activities of Girls, Boys and Adolescents (MANNyA)16 that was included as part of the Annual Urban Household Survey (EAHU, by its Spanish initials) during 2012. The MANNyA was carried out on the basis of an inter-institutional initiative between the Ministry of Labor, Employment and Social Security, the National Secretariat for Children, Youth and Family, the National Commission for the Eradication of Child Labor, the National Institute of Statistics and Censuses (INDEC, by its Spanish initials) and the Provincial Statistical Offices.\n\nThe sample consisted of children and adolescents aged 5 to 17 from urban conglomerates in Argentina. It was a probabilistic, stratified and multi-stage sample of 34,487 households, with a total of 25,915 children and adolescents surveyed16.\n\nThe data from the MANNyA secondary database16, the basis of this analysis, was collected using a self-administered instrument. The databases of MANNyA 2012 are public and can be obtained in the web site of INDEC.\n\nThe instrument included closed-ended questions aimed at obtaining what children and adolescents usually do in their leisure time.\n\n\"Healthy use of leisure time\", defined as the time for rest and leisure activities, play and recreational activities suitable for the age. It also implies the right to participate freely in cultural life and the arts7. In the framework of the analyzed module, leisure time activities are defined as everything that the child does before or after school, or on the weekends16.\n\nThe dimensions of analysis for this study were:\n\n- School activities (homework or studying for school);\n\n- Sports/physical recreation (football, swimming, cycling/horseback riding, etc.);\n\n- Art (painting, theatre, music, dance, language or art-related workshop or course);\n\n- Socialization (going out with friends to the cinema, to the square, to the cyber cafe, etc.);\n\n- Use of Information and Communication Technologies (ICT; computer or netbook); and\n\n- Internet use\n\n\"Social vulnerability\", defined as the situations of insecurity and defenselessness experienced by communities, families, and individuals in their livelihood conditions, as a consequence of the impact caused by any socio-economic event. In addition, the management of resources and the strategies used by them to cope with the effects of this event were considered17. Today, social vulnerability is considered one of the main social determinants of health10, where most health problems can be attributed to people's socio-economic conditions.\n\nFor the construction of this variable, a Social Vulnerability Index (SVI) was prepared (Table 1) based on the data from the MANNyA secondary database16. The dimensions and weighting values of the conceptualization of social vulnerability were based on previous publications17,18.\n\n* It represents the ratio between the total number of people in the household and the total number of rooms or spaces available in the household (excluding bathroom(s) and kitchen(s)(Instituto Nacional de Estadísticas y Censos).\n\nThe predominant materials of the constituent components of the dwelling (floors, walls, and roofs) are assessed and categorized in relation to their strength, resistance, and capacity for thermal, waterproof, and sound insulation. The dwellings are classified as:\n\nCalmat I and II: the dwelling was made from resistant and solid materials used in floors and roofs, although it may/ may not incorporate insulating or finishing elements in at least one of these. Mosaic/tile/wood/ceramic/carpet floors were considered. The roof had a membrane/asphalt singles and did have a ceiling/interior cladding.\n\nCalmat III: the dwelling was made from resistant and solid materials used in floors and roofs, but it lacks elements of insulation or finishing in all of these. Cement floor/fixed brick floor was considered. Roof with tile/slab roof without any cover, slate/clay tile, and no ceiling/interior cladding.\n\nCalmat IV and V: the dwelling was made of non-resistant materials in at least one of the constituent components. Loose brick/earth floor and roofs of sheet metal without cover, fiber cement/plastic sheet, corrugated roofing sheet, cane/board/straw with mud/ straw alone were considered and there was no ceiling/interior cladding.\n\n*In the weighting structure, greater importance has been given to the occupation dimension (dependency burden of income earners), given that the association of the population with the labor market becomes a key factor in social vulnerability, and monetary income can change the situation of social inclusion/exclusion more immediately.\n\nThe categories included material assets, such as employment and housing, and non-material assets, such as those related to human capital (access to the health system and educational system of the head of household).\n\nThe construction of the SVI was based on the selection of dimensions represented by different categories which, depending on the risk situation, were defined as «moderate» or «critical».\n\nSince each of the selected categories may have different levels of intensity, it was decided to define differential weights within them18.\n\nNext, the SVI of each household where the child or adolescent is living was categorized as follows: without SVI was assigned to those cases with a value of 0.00; low or moderate SVI was assigned to those cases with values between >0.00 and ≤0.45; high SVI was assigned to those cases with values greater than 0.45.\n\nThe variables of interest were analyzed descriptively using position and dispersion measurements and frequency distribution.\n\nGiven the hierarchical structure of the data (individuals grouped in regions of the country), a multilevel logistic regression analysis was conducted to explore the relationship between the social vulnerability and the healthy use of leisure time. The models considered socio-economic and socio-demographic variables at two levels: an individual level, relating to the child/adolescent (performance of domestic and economic activities, gender, age, school attendance, health coverage, illiteracy), and a household level (level of education of the head of household). An empty model (Model 0) was performed, and the level 2 variance (household) and the intraclass correlation coefficient (ICC) were calculated.\n\nFirstly, a univariate analysis was carried out and, from the variables that resulted in statistical significance, a multivariate model of random intersection was constructed by introducing one variable at a time. The standard errors were calculated taking into account the cluster effect of each region of the country. The variable-addition models were compared using the likelihood ratio test. The proportion of the variance at level 2 explained (PVE) by the different models was calculated as PVE= (V) × 100.\n\nThe statistical package Stata® v14.2 (Stata Corporation, College Station, Texas, USA) was used for all the analyses, and a p<0.05 value was considered statistically significant.\n\nThe study was carried out on the basis of a secondary analysis of the MANNyA database16, compiled by different public bodies under the leadership of the INDEC. In Argentina, public statistics produced by the State are part of the National Statistical System created by Law No. 17622, which guarantees confidentiality and the Protection of Personal Data through Law No. 25326.\n\nThe database is currently public and open-access, and is de-identified by the responsible public body19. Thus, this study did not require an evaluation by an ethics committee and, in addition, qualifies for the status of being exempt from obtaining informed consent.\n\n\nResults\n\nAs shown in Table 2, the sample consisted of 52.6% males and 63.5% adolescents. In total, 32.2% of the respondents were from Gran Buenos Aires and 30.9% from the Pampas region. It is also observed that 95.5% were attending school at the time of the survey and 58.1% had some kind of health coverage.\n\nValues expressed in proportions (%).\n\nSchool Attendance of the child/adolescent defined as: self-report of attending school at the time of the survey.\n\nHealth Coverage of the child/adolescent defined as: health insurance (including Comprehensive Medical Attention Program (PAMI), mutual/prepaid/emergency service). Public plans and insurance were not considered as coverage.\n\nChild/adolescent Does Not Know How to Read or Write defined as: self-reporting of not knowing how to read or write at the time of the survey.\n\nSocial Vulnerability Index (SVI) of the child/adolescent defined as: The SVI of each household where the child or adolescent is included according to the categories (Without SVI: value of 0.00/SVI low/moderate: >0.00 and ≤0.45/SVI high: >0.45).\n\nLife Stage of children/adolescents defined as: childhood <10 years old and adolescence ≥ 10 to 19 years old according to the WHO classification.\n\nData Weighted by age and gender stratum for each city to represent the general population.\n\nTable 3 shows the prevalence of activities of healthy use of leisure time, according to socio-demographic characteristics. It was observed that the prevalence of performance of school activities was 90.1%, higher in the group with health coverage (91.9%) and in the literate group (92.6%). Art activities (21.8%) had the lowest prevalence of performance as compared to the other activities of free-time use.\n\nSE: Standard Error.\n\n95%CI: 95% Confidence Interval.\n\nValues expressed in proportions (%).\n\nLife Stage defined as: childhood <10 years old and adolescence ≥ 10 to 19 years old according to the WHO classification.\n\nRegion of the child/adolescent defined as: Greater Buenos Aires, Cuyo, Northeast, Northwest, Pampa, and Patagonia.\n\nSchool Attendance of the child/adolescent defined as: self-report of attending school at the time of the survey.\n\nThe socialization, sports/recreational activities, use of ICTs and the Internet showed an intermediate prevalence between 51.1% and 74.2%, being, in most cases, higher in males than in females. Also, lower values are observed in groups that do not attend school, do not have health coverage and are illiterate.\n\nTable 4 shows the main results of the subgroup analysis. In the group without SVI (10.9% of the sample), it was observed that the highest prevalence was the performance of school activities (90.9%), with the art activities being the least prevalent (19.2%).\n\nSE: Standard Error.\n\n95%CI: 95% Confidence Interval.\n\nValues expressed in proportions (%).\n\nSocial Vulnerability Index (SVI) of the child and adolescent defined as: The SVI of each household where the child or adolescent is included according to the cohort categories of: Without SVI: value of 0.00/low/moderate SVI: >0.00 and ≤0.45/ high SVI: >0.45.\n\nLife Stage defined as: childhood <10 years old and adolescence ≥ 10 to 19 years old according to the WHO classification.\n\nRegion of the child and adolescent defined as: Greater Buenos Aires, Cuyo, Northeast, Northwest, Pampas, and Patagonia.\n\nData Weighted by age and gender stratum for each city to represent the general population.\n\nHealth Coverage of the child and adolescent defined as: health insurance (including Comprehensive Medical Attention Program (PAMI), mutual/prepaid/emergency service). Public plans and insurance were not considered as coverage.\n\nChild/adolescent knows how to read or write defined as: self-reporting of not knowing how to read or write at the time of the survey.\n\nSocial Vulnerability Index (SVI) of the child and adolescent defined as: The SVI of each household where the child or adolescent is included according to the cohort categories of: Without SVI: value of 0.00/low/moderate SVI: >0.00 and ≤0.45/ high SVI: >0.45.\n\nData Weighted by age and gender stratum for each city to represent the general population.\n\nAmong those with a low or moderate SVI (87.1%), the distribution of the performance of leisure time activities was the same as that of the group without SVI, although with higher values in the cases of ICTs (75.1%), Internet (66.7%) and art activities (22.4%).\n\nIn the group composed of those with a high SVI (2.1%), the distribution of the performance of leisure time activities changes as compared to the other two groups of SVI. While school activities (83.1%) and art activities (11.4%) continues to be the most and least popular choice, respectively, sports/recreational activities are in second place (48.1%), replacing ICTs, which have moved into third place (45.1%). In turn, socialization activities (36.3%) were more prevalent than Internet use (33.7%).\n\nIn the multilevel models, statistically significant associations between some leisure time use activities and social vulnerability are seen (Table 5).\n\nMultilevel logistic regression model (n= 25,915). Module on Activities of Girls, Boys and Adolescents, Annual Urban Household Survey, 2012.\n\nOR: (odds ratio) obtained by a logistic regression model, where the dependent variable was the leisure time use activities (such as dichotomous).\n\np: level of statistical significance <0.05.\n\nSE: Standard Error; ICC: Intraclass Correlation Coefficient.\n\nSocial Vulnerability Index (SVI) defined as: The SVI of each household where the child or adolescent is included according to the cohort categories of: Without SVI: value of 0.00/low/moderate SVI: >0.00 and ≤0.45/high SVI: >0.45.\n\nHealthy Use of Leisure Time Activities: as a dichotomous variable (yes/no) (school, sports/recreational, art, socialization, ICT and Internet).\n\nRegion defined as: Greater Buenos Aires, Cuyo, Northeast, Northwest, Pampas, and Patagonia.\n\nIndividual variables:\n\nHousehold activities of the child/adolescent defined as: activities carried out in the home in an intensive and/or non-intensive manner;\n\nEconomic activities of the child/adolescent defined as: worked in the reference week and/or worked during the last year.\n\nAge defined as: childhood <10 years old and adolescence ≥ 10 to 19 years old according to the WHO classification.\n\nSchool Attendance of the child/adolescent defined as: self-report of attending school at the time of the survey.\n\nHealth Coverage of the child/adolescent defined as: health insurance (including Comprehensive Medical Attention Program (PAMI), mutual/prepaid/emergency service). Public plans and insurance were not considered as coverage.\n\nChild/adolescent Knows How to Read or Write defined as: self-reporting of not knowing how to read or write at the time of the survey.\n\nContextual variables:\n\nHead of Household’s Education Level defined as: incomplete elementary education (including special education), complete elementary education, incomplete high school education, complete high school education, incomplete college education, complete college education, and without schooling.\n\nSex of Household Head defined as: male and female.\n\nEmployment Status of the Household Head defined as: employed, unemployed, inactive, less than 10 years.\n\nModel 0: empty model only of random intersection of the dependent variable attributable to the region.\n\nModel 1: bivariate analysis between healthy leisure time activities and the SVI.\n\nModel 2: individual multivariate. Model 1+ all individual variables such as child/adolescent’s household activities (intensive or non-intensive) and child/adolescent’s economic activities (worked in the reference week or worked during the last year) sex of the child/adolescent, age of the child/adolescent, current school attendance of the child/adolescent, health coverage of the child/adolescent, child/adolescent can read and write with random intersection.\n\nModel 3: multivariate multilevel final model: all individual and contextual variables with random intersection.\n\nIn the multilevel model, the non-performance of school activities was statistically associated with low/moderate SVI, with an OR of 1.165 (p=0.023).\n\nIn analyzing the relationship between high SVI and the risk of not performing sports/recreational activities, it was observed that the OR was 1.292, although this association was not significant (p=0.088).\n\nWith respect to the relationship between high SVI and the non-performance of art activities, an OR of 2.232 was found; that is, a higher risk of not performing these activities, being statistically significant (p<0.001). This was similarly reflected in the relationship between SVI in the highest stratum and the socialization activities (OR 1.999 and p=0.002).\n\nWith respect to the relationship between high SVI and the non-use of ICTs, a statistically significant association was observed, in which the OR was 14.171; that is, high SVI resulted to be a risk factor (p<0.001). On the other hand, it could be observed that the non-use of the Internet during leisure time in the group with high SVI yielded an OR of 21.887, being significant (p<0.001).\n\nBased on the aforementioned, in the final models (Model 3), which considered individual and contextual variables, a clear upward gradient in the likelihood of unhealthy use of leisure time is observed when there are increased SVI levels in the cases of art, socialization, ICT, and the Internet.\n\n\nDiscussion\n\nOur results highlighted the impact of a high social vulnerability index on the reduced performance of some healthy leisure time activities such as art, socialization and Internet use, among children and adolescents in Argentina.\n\nA new look at the concepts of leisure time in Latin America has to be constructed, rescuing the profound knowledge and practices already elaborated in Latin American territories20. However, reflection on this relationship requires various perspectives and levels of analysis. In this sense, it is necessary to highlight the influence of the public social protection policies in guaranteeing the right to leisure time in childhood and adolescence.\n\nSome risk factors from the social sphere where biographies of children and adolescents are developed may have an impact on the ways in which they perform in society. The fact that a significant part of the risk to their health and quality of life occurs in this context was highlighted in the field of social epidemiology21.\n\nAs Feito22 suggests that vulnerability has a dimension of susceptibility to harm, conditioned by intrinsic and extrinsic factors, anchored in the radical fragility of the human being, but undoubtedly largely attributable to social and environmental elements. In this sense, our study showed that a significant part of the population under analysis had some degree of social vulnerability, which makes us consider this indicator as one social determinant of health10.\n\nRegarding the healthy use of leisure time in the population under analysis, it was observed that some activities were more prevalent than others; generally, art, sports/recreational, and socialization activities were less frequent. This information is important because, as other authors have shown23,24, these activities should be proposed as strategies to promote the health of children and adolescents at social risk25, since they contribute to the psychosocial and physical state of children and adolescents, while at the same time promoting life skills. This fact is relevant since they could also be the activities to be promoted through different public health policies and programs in the country.\n\nIn addition, some activities such as sports and recreation were performed only by half of the sample. This leads to the need to promote these activities in order to have a positive impact on some aspects related to the physical and mental health and nutrition of children and adolescents in a complex epidemiological context, characterized by the increase in chronic diseases, low physical activity, and malnutrition26,27.\n\nAnother aspect to highlight is the high prevalence of school activities during leisure time. This can be explained by the implementation of public policies of great impact in the years prior to this survey, such as the Universal Child Allowance (AUH), in force since 200928 and considered a key element for staying at school.\n\nIn this sense, the debate on the psychosocial development in childhood and adolescence should be focused not only on school activities, but also on the possibility of choosing and performing multiple leisure time activities freely; activities that entail an enjoyment linked to them. In this sense, we agree with what Fredriksson and colleagues stated in a recent publication, in which they suggest that increasing the participation of young people in leisure activities, especially those from more socially vulnerable environments, can help to reduce social inequalities in health. In this sense, it is necessary to promote varied activities, both structured and unstructured, during free time29.\n\nAnother aspect to consider is that of gender differences in the performance of activities during leisure time. For example, it is observed that “sexual division in the activities between men and women is already established in childhood and adolescence”30. Our results showed that men tended to do more sports activities and women more art activities.\n\nWith respect to some social determinants of health, such as literacy or school attendance, in all the activities the prevalence was found to be higher in the low and without SVI subgroups, highlighting the weight of these determinants. In this sense, the ecological study conducted by Viner et al. on the health of adolescents suggested that the most effective health interventions are probably those addressing structural changes, e.g. access to education31.\n\nFurthermore, it should be noted that the use of digital-free time, mainly focused on the use of the Internet and ICTs, has become increasingly present in the activities of children and adolescents and has become an agent of socialization32.\n\nHowever, our results show that the use of the Internet is significantly conditioned by social vulnerability. In this sense, it was observed that, in the group with the highest level of vulnerability, there is a 14 times higher risk of not using the Internet. This is relevant and shows how the condition of vulnerability becomes a determinant barrier when accessing certain goods and services, especially if progress is to be made in reducing digital divides33.\n\nOur findings on the use of ICTs were similarly, albeit less strongly, reflected in the use of the Internet; that is, it was observed that there was a risk of not using ICTs in the groups with high levels of social vulnerability. It should be noted that two years before this survey, Argentina implemented the “Connecting Equality” policy34, a federal broad-scope program. This is important because the effectiveness of the digital divide reduction policies, especially among the most vulnerable groups, could present certain barriers to access35.\n\nThere were also marked differences with respect to art and socialization practices, where it was found that the higher the social vulnerability index, the higher the risk of not performing these activities. However, this result highlights the importance of promoting this type of activity in children and adolescents, since, according to the evidence and based on the findings by Wald36, the changes perceived as a result of participating in art workshops are closely linked to feelings of well-being, the development of personal capacities and the strengthening of group relations, which may be linked in a broad sense to the paradigm of health promotion.\n\nFurthermore, the subjective processes of socialization in human health acquires a central role. In this line, the World Health Organization37 hierarchized and gave relevance to these aspects within the concept of quality of life. Several points can be highlighted in our results and our contributions to the knowledge of the situation of leisure time use in children and adolescents.\n\nFirstly, it was found that in 2012 there was a social inequality gap, as measured by the SVI. On this point, and going back to the theoretical framework that underpinned this proposal, it is necessary to reflect on the weakening of social networks, unfavorable economic scenarios and the differential impact of targeted social protection policies at national level38.\n\nSecondly, the negative effect of social vulnerability on some specific activities was highlighted, such as those related to art, socialization processes and access to goods and services such as the Internet. From here, it is possible to postulate that this would not be fully contributing to the achievement of the Rights of the Child8, which claim the free participation in cultural life and the arts7.\n\nThirdly, it was observed that, in a large part of the activities of healthy leisure time use, the presence of social vulnerability conditions its full performance; in this sense, it could be believed that these activities are hampered or compete with other activities e.g. those that are domestic and economic30.\n\nIn a study carried out in Ecuador, Mexico and Peru, CEPAL and UNICEF stressed that work, both paid and unpaid, is another activity that occupies an important part of adolescents' time; this reality is not in line with the fundamental rights of this group30. Thus, we face a double challenge concerning the effective right to the healthy use of leisure time. On the one hand, the aim is to promote leisure and welfare activities for children and adolescents30; sports, art, and socialization activities. On the other hand, it is necessary to consider the digital divide and access to differential goods and services, which could be considered as socialization agents32 in this group.\n\nAnother weakness of the proposal is that the research question was addressed based on the analysis of a secondary data source, so it is possible that some aspects related to the construction of the SVI were left out. However, the methodology for the construction of this index is flexible and there is not a sole theoretical frame of reference. Lastly, another limitation was the data collection date (2012), where some aspects may have been modified so far.\n\nHowever, this study has many important strengths. Among them, the large sample size stands out. It allowed for multivariate analyses and adjustments by multiple confounders and the national representativeness of the sample, since the survey of the secondary database was coupled to the Annual Urban Household Survey (EAHU) by following the application of a rigorous probabilistic sampling, thus ensuring the accuracy of the data obtained and the scope of the entire urban population of the country16.\n\n\nConclusions\n\nIn conclusion, the presence of social vulnerability has an unfavorable impact on the performance of some healthy leisure time activities, such as art, socialization and use of the Internet in the group of children and adolescents in Argentina. The obtained findings lead us to highlight some strategic moves concerning the field of health social determinants where the SVI could be a useful tool to guide health promotion initiatives in the population of children and adolescents.\n\n\nData availability\n\nThe MANNyA data and documentation are available at https://www.indec.gob.ar/bases-de-datos.asp?solapa=7.", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nLarson RW, Verma S: How children and adolescents spend time across the world: work, play, and developmental opportunities. Psychol Bull. 1999; 125(6): 701–36. PubMed Abstract \| Publisher Full Text\n\nEccles JS, Templeton J: Chapter 4: Extracurricular and Other After-School Activities for Youth. Review of Research in Education. 2002; 26(1): 113–80. 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Reference Source\n\nSalvia A: Argentina 2007: condiciones de vida de la niñez y adolescencia. Una mirada desde la perspectiva de los derechos. Informe Nº 2 del Barómetro de la Deuda Social de la Infancia. Observatorio de la Deuda Social Argentina de la Universidad Católica Argentina. Buenos Aires: UCA; 2008. Reference Source\n\nTuñon I, Salvia A: La deuda social con la niñez y adolescencia: magnitud, evolución y perfiles. Observatorio de la deuda social argentina. Pontificia Universidad Católica Argentina; 2010. Reference Source\n\nLeversen I, Torsheim T, Samdal O: Gendered leisure activity behavior among Norwegian adolescents across different socio-economic status groups. 2012; 3(4): 355. Publisher Full Text\n\nInstituto Nacional de Estadística y Censos de la República Argentina: Módulo de niñas, niños y adolescentes (MANNyA). Buenos Aires: INDEC; 2012; [cited 2017 December 12]. Reference Source\n\nPizarro R: La vulnerabilidad social y sus desafíos: una mirada desde América Latina. Santiago de Chile: CEPAL; 2001. Reference Source\n\nCon M, Susini S, Catalá S, et al.: Índice de Vulnerabilidad Social (IVS) Documento metodológico. Buenos Aires: Ministerio de Educación Del GCBA. 2011. Reference Source\n\nInstituto Nacional de Estadísticas y Censos de la República Argentina: Actividades de niñas, niños y adolescentes. Buenos Aires: INDEC; 2012; [cited 2017 December 12]. Reference Source\n\nElizalde R, Gomes C: Ocio y recreación en América Latina: conceptos, abordajes y posibilidades de resignificación. Polis. 2010; 9(26): 19–40. Publisher Full Text\n\nMunist M, Santos H, Kotliarenco MA, et al.: Manual de identificación y promoción de la resiliencia en niños y adolescentes. Washington: OPS/OMS; 1998. Reference Source\n\nFeito L: [Vulnerability]. An Sist Sanit Navar. 2007; 30 Suppl 3: 7–22. PubMed Abstract \| Publisher Full Text\n\nTuñón I, Laiño F, Castro H: El juego recreativo y el deporte social como política de derecho: Su relación con la infancia en condiciones de vulnerabilidad social. Educación Física y Ciencia. 2014; 16(1). Reference Source\n\nKrumm G, Lemos V: Actividades artísticas y creatividad en niños escolarizados argentinos. Int J Psychol Res. 2012; 5(2): 40–8. Reference Source\n\nMoreira González A, Murillo Gamboa P: Habilidades para la vida como estrategia de promoción de la salud en niños y niñas en riesgo social: programa educativo de enfermería. Enfermería Actual en Costa Rica. 2016; 30. Publisher Full Text\n\nOrganización Mundial de la Salud: Actividad física. OMS; 2018; [cited 2018 May 25]. Reference Source\n\nMinisterio de Salud de la Nación Argentina: Encuesta Nacional de Nutrición y Salud. Documento de resultados. 2007; 182. Reference Source\n\nAdministración Nacional de la Seguridad Social de la Nación Argentina: Asignación Universal por Hijo. ANSS; 2018; [cited 2018 May 25].\n\nFredriksson I, Geidne S, Eriksson C: Leisure-time youth centres as health-promoting settings: Experiences from multicultural neighbourhoods in Sweden. Scand J Public Health. 2018; 46(20_suppl): 72–9. PubMed Abstract \| Publisher Full Text\n\nComisión Económica para América Latina y el Caribe & Fondo de las Naciones Unidas para la Infancia: El derecho al tiempo libre en la infancia y adolescencia. Santiago, Chile: CEPAL/UNICEF; 2016. Reference Source\n\nViner RM, Ozer EM, Denny S, et al.: Adolescence and the social determinants of health. Lancet. 2012; 379(9826): 1641–52. PubMed Abstract \| Publisher Full Text\n\nPallarés Piquer M: Medios de comunicación: ¿espacio para el ocio o agentes de socialización en la adolescencia? 2013; 231. Publisher Full Text\n\nFondo de las Naciones Unidas para la Infancia: El estado mundial de la infancia 2017. Niños en un mundo digital. New York: UNICEF; 2017. Reference Source\n\nMinisterio de Educación y Deportes de la Nación de la Nación Argentina: Conectar Igualdad. [cited 2018 May 25]. Reference Source\n\nArcos E, Sanchez X, Toffoletto MC, et al.: Social protection systems in vulnerable families: their importance for the public health. Rev Saude Publica. 2014; 48(3): 398–405. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWald G: Promoción de la salud a través del arte: estudio de caso de un taller de fotografía en \"Ciudad Oculta\", la villa Nº 15 de la Ciudad de Buenos Aires. Salud colectiva. 2009; 5(3): 345–62. Publisher Full Text\n\nOrganización Mundial de la Salud: Que es calidad de vida? Grupo de la OMS sobre la calidad de vida. Ginebra, Suiza: OMS; 1996. Reference Source\n\nRuscheinsky A, Martínez D: Riesgos sociales en América Latina y políticas publicas: actores políticos y prácticas operativas. Interações (Campo Grande). 2014; 15(1): 121–33. Publisher Full Text" }	[ { "id": "40064", "date": "27 Nov 2018", "name": "Suzana Alves de Moraes", "expertise": [ "Reviewer Expertise Epidemiology", "Statistical methods applied to Epidemiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGeneral Comments: The study aims to investigate the association between Social Vulnerability Index (SVI) and healthy use of leisure time (outcome). The authors applied multilevel analysis including in the models SVI and individual variables potentially related to the outcome. In general, the manuscript is reasonably well written (exception the section Discussion), and some specific questions, listed below, claim to be replied.\n\nSpecific Comments:\n\n1. Abstract: The Abstract is structured, although if taking into consideration the specific comments listed forward, the Abstract might be re-written. The keywords might include: ‘multilevel analysis’ and ‘leisure time’ instead of recreation.\n\n2. Introduction: This section is adequately formatted, although it should be opportune to include 2 or 3 papers which have identified similar or different associations, in order to reinforce the present purpose.\n\n3. Methods: 3.1. Response rates in different multi-stage sampling process should be reported. On the other hand, non-respondent characteristics should be shown, related to participants. Moreover, it is important to ask if the authors had tested interactions between potential differences in any variable and the main variable (SVI) (participants versus non-participants) related to the outcome to evaluate potential selection bias [See Szklo & Nieto: Epidemiology- beyond the basics- 2nd Edition, 2007]. Weighting variables to correct ‘design effect’ (multi-stage sampling) has been considered? It should be clear.\n\n3.2. How the authors could be sure that a self-administered Instrument in that population corresponds to their reality? Perhaps it should be relevant to replicate some information among, at least, 10% of the population and calculate the intraclass correlation coefficient to evaluate reliability. If this procedure or a similar one has been considered to that evaluation it might be important to refer it.\n\n3.3. The term ‘Result Variable’ should be rewritten as “Outcome”.\n\n3.4. SVI- The variable was classified as ‘No risk’, ‘Low/Moderate’ and ‘High risk”, so those 3 strata should be described in the 4th paragraph of the explanatory variables (page 4).\n\n3.5. In Table 1 (Method section), why different categories into the same dimension have got equal weight? Ex:, To the dimension Occupation, different categories has been attributed the same weight (0,30). This comment seems to be reasonable, mainly because the authors reported (last footnote under Table 1) the great importance of this variable, in terms of its discrimination power (‘a key factor’).\n\n3.6. Statistical Analysis: In complex sampling (multistage), odds ratios use to be overestimated, being preferable to use prevalence ratios. It seems to be recommended to comment about that. How many levels has been considered in the multilevel analysis? What variables composed the level 2: Region or households? It is not clear. The cluster effect of Region, doesn't seem to be estimated (where are ICC values for Region?). The formula PVE =V x 100 might be better explained, it doesn't seem to be clear. If Region was not considered as an ecological variable in the analysis (level 2 or 3) it is not plausible to show prevalence according Regions in the descriptive analysis. What References related to multilevel analysis were used?\n\n4. Results: 4.1. Table 2: In classifying the variable: Knows how to read and write as yes/no, it is recommended to review the results since they seem to be inverted in relation to the definition shown in footnote.\n\n4.2. Tables 3 e 4: The information on those tables are heavy and it is redundant to show SE and CI. Perhaps SE should be suppressed. Please, review the results related to the variable: Knows how to read and write classified as yes/no (Table 3). Why Region has been included, since the group of variables shown here are individual variables? See comment referred on item 3.6. (statistical analysis). In Table 3, there is no mention related to weighted data, why?\n\n4.3. Table 5: a) In general, odds ratios showed higher magnitudes than expected. Probably, as referred above, it is a result of using odds ratios instead of prevalence ratios. Moreover, the large confidence intervals denote lack of precision, due to the strata dilution (high SVI category). On the other hand, multicollinearity between SVI and other contextual variables shown on the footnote may contribute to that. It might be a good strategy to join moderate + high strata as one. b) ICC values are highest than expected and they increase from model 0 to model 3. How does it is plausible, since after adjusting by individual variables, ICC should decrease because the influence of the last one variables? c) By the way, those contextual variables has not been described in the sections ‘Methods’ and/or descriptive ‘Results’, as well as some individual variables like household activities of the child/adolescents, etc… d) One strategy to evaluate multicollinearity is to run matrix correlations among the independent variables. e) It is recommended to add the model quality adjusted indexes (Hosmer & Lemeshow χ2 in the case of logistic regression models or likelihood ratios if prevalence ratios are used).\n\n5. Discussion: Most paragraphs on this Section are not in consonance with the study purposes and results. Some of them are only related to the descriptive analysis (Ex: paragraphs 6 e 7). There were neither specific discussion related to the multilevel analysis nor comparison between such results and those found in the literature. Some paragraphs are extremely far from the supposed aims of the present study (Ex:, paragraphs 2, 5, 8, 10,11, 12). In paragraph 4 the authors reported: … “in this sense, our study showed that a significant part of the population under analysis, had some degree of social vulnerability, which makes us consider this indicator as one social determinant of health”. Based on the text highlighted above, it should be emphasized that just because most of the population (around 90%) were classified as moderate or high SVI, this variable probably does not keep good discrimination power, so that it should not be considered as a good determinant of health. There is a statement that is not found in the respective Table 5 (in paragraph 12, the authors reported an odds ratio = 14. This value was not found in Table 5, and its interpretation is misleading, since odds ratio is not risk). Strengths and limitations reported in paragraphs 21 and 22 were poorly described. The main limitation is not the study base (secondary) but mainly related to the representatives what was not reported by authors (missing data in the sample process could lead to selection bias), and the ‘self-administered instrument’ without mention about who replied the questions (parents, children, adolescents) could lead to measurement bias. No statement related to potential selection or measurement bias was reported by authors. The conclusions are equally misleading. Taking into consideration the scope of this section, the authors did not present a consistent discussion, and, so, the section should be completely re-written.\n\n6. References: As already reported by Reviewers, no relevant references related to the analytical phase (multilevel analysis) were found. Among 38, 15 References are related to International Institutions, or similar. There should be specific references related to the study aims and the main findings.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "40066", "date": "27 Jun 2019", "name": "Myriam Guerra-Balic", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGeneral comments: This manuscript studies the relationship between social vulnerability index and healthy use of leisure time. The manuscript can be considered as a case study, as it focuses only on the Argentinian population. Some information is missing in order to understand better what authors would like to show, and they also need to improve the interpretation of the results obtained at the discussion section. Please, find my specific comments that can help authors.\n\nSpecific comments: Abstract: Even it is known what ICT means, I suggest to add what it means in words.\n\nIntroduction: This section can be considered somehow weak. Concerning schools, it is missing information about how school schedule is in Argentina, for example, how long children are staying at school daily; is it the same schedule for primary, secondary and high school? Do they have all afternoon free, or some of them? Do they have more than one turn (morning turn and afternoon turn? It could be interesting to know about public/private schools, because sometimes their schedule is different, and if some activities are included into the daily schedule. In fact, the survey asks about the timetable they have at school, but has not been considered. Updated data from other countries (not only Latin American) are missing, just to compare and discuss later about them. It could be of interest to conceptualize what health is, and how leisure time can be healthy, not only physically (aerobic, strength, body composition, etc), but also from a functional, psychological and social point of view.\n\nMethods: It could be interesting to explain more about how data collection was done. Searching the original survey[spanish] it is understood that it is not intended to know about leisure time (only one block of questions, of a total of 28, asked about it), but to know about their working activities. Perhaps, because of that, this information is not enough to interpret when analysing data. It is suggested, as well, to annex the survey, even it is in Spanish. If it is a self-administered survey, all the questions were the same for all the children and adolescents (5-17 years old)? If there were the same, for a 5 year old child it can be difficult to answer some (or all) the questions, so it is needed to know who helped them to answer. Moreover, authors presented a number of participants that did not know how to read and write, so who answered? In fact, the item 29 of the survey asks about the adult’s participation when answering, but, was a parent, older brother/sister, relative, baby sit…? Was there an age cut-off point considered when receiving support for answering? Did the survey taker participate, as well? All these aspects make the methods confusing for getting the data. Was it really a self-administered survey or there was a survey taker? As the data studied were obtained from the 2012, co-authors should state if there was or not a later survey that explores leisure time. More than 5 years could give different results. In fact, in the website, there is another survey applied during 2016-2017 obtaining data concerning children’s and adolescent’s work activities (https://www.indec.gov.ar/ftp/cuadros/sociedad/eanna_2018.pdf)[Spanish] Social Vulnerability Index: Why the dimensions Overcrowding and Occupation have the same weighting? In Table 1 it is suggested to use the same format when referring to Calmat (use Roman numbers in the table and in the notes) No information about regions is given in Methods, when later it is included in results. Why? If considering Regions, it would be necessary to describe better each region: number of population, number of children, socio-economic level of the region. I suggest to present the methods with a flowchart, so it could be easier to understand what methods authors applied and.\n\nResults: Table 2: Why are there children not attending schools? Was it because illness, or education at home, or need to work and earn money, or other? Are correct the results given related to knowing how to read and write? Could it be a confusion and the numbers are changed? Did authors control by members or number of children/adolescents in each family? Why public health plans and insurance were not considered? All the children and adolescents have it by law? When talking about performing sport/recreational activities, were they structured, or simply playing at home/neighbourhood involving physical activity? Were they paid or not? When authors stated that: “our study showed that a significant part of the population under analysis had some degree of social vulnerability, which makes us consider this indicator as one social determinant of health”, couldn’t it be a bias?\n\nDiscussion: This section can be improved, taking into account all the suggestions about information to be added. More structured text is necessary for understanding what authors wanted to show. Sometimes the information is mixed, and it is confusing. This can take to weak conclusions, not giving properly answers to the objectives proposed. Limitations have been considered, but not clear enough, and the proposals for future studies are missing.\n\nReferences: In general, references are limited. First, they should be more updated. And if updated, data obtained from could not match with the period the survey was done. For example, reference number 28, from year 2018, does it give information about the Asignación Universal por Hijo during 2012? If it is not, authors should discuss it. Many other references are simply informative, based on websites (most of them official ones). When I consulted some of these websites (for example reference 4 and 34), I did not find information about when the data were obtained, so it might be difficult to interpret the results comparing the survey of 2012. Please, find several papers suggested for improving the content of this manuscript, even authors can find other ones for sure.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	2	https://f1000research.com/articles/7-1619
https://f1000research.com/articles/10-272/v1	06 Apr 21	{ "type": "Research Article", "title": "Motoda Nagazane’s standards of Confucianism – between Confucianism and Emperor Centralism", "authors": [ "Seogin Eom" ], "abstract": "This paper discusses the ideological significance of the activities of Motoda Nagazane who, in the latter half of his life, became an attendant of Emperor Meiji as a member of the Kumamoto school of practical science. Whilst there were trends towards modernisation and Westernisation, Motoda Nagazane led a conservative reaction attempting to restore Confucianist politics/policies. I scrutinise the theories of revolution and lineage considering the history of East Asian Confucianism and comparing Motoda’s assertions to the views expressed by Kumazawa Banzan. In doing so, I assert that Motoda’s consistent attitude shows that he does not approve of the theory of revolution and that he regards the theory of lineage as an established fact. Thus, he highlights the cultivation of virtues in rulers, adopting the stance typically taken by Confucian scholars in the history of Japanese ideology. In ‘Lessons of the Emperor’s Way’, Motoda attempts to support the meaning of ‘The Three Sacred Treasures’ through Confucian texts. My evaluation of this text results in the view that in this discourse, Motoda transcends the significance of harmonising the deep and difficult ‘Lessons of the Emperor’s Way’ with Confucianism, which is easy to impart. I deduce that Confucianism was positioned above all else as the absolute/comprehensive standard in Motoda’s thought and that his endorsement of the Emperor’s way was proscribed within the ideological boundaries of Confucianism. Through the above analysis, I conclude that Motoda was an anachronistic Confucian scholar who truly endeavored to realise the kingship politics of Yao and Shun in the early Meiji era. While it is acknowledged that he was lagging behind his contemporaries, it is shown that this seemingly backward stance emanated from his serious Confucian scholarship. Further, his assertions differ from the plain-spoken Confucianist Emperor centralism that emerged in later years.\n本稿は、熊本実学派の一員として人生の後半期に明治天皇の側近となり、当時の西洋近代化の流れにあって、いわゆる保守反動の儒教主義的な政治・政策の復活を主導した、元田永孚の活動の思想的意義を論じたものである。革命論と血統論の検討においては、元田の主張を熊沢蕃山との比較、さらに東アジア儒学史に照らしてみることで、革命論を是認せず血統論を既定の事実として、君主の君徳培養に強調したのが元田の一貫した姿勢であり、それは日本思想史における典型的な儒学者の立場を示すものであったことを論じた。「皇道の訓解」の検討では、「三種の神器」の意味を儒教テキストによって裏付けようとする元田の言説を対象として、それは奥深くて知り難い皇道を教えやすい儒教によって補完するという意味を越えて、元田の思考では第一に儒教という絶対的・包括的なスタンダードがあり、その限りにおいて皇道の意義づけを行っていた、という見解を提示した。以上の検討を通して、元田は堯舜の王道政治を明治前期において本気で実現しようと臨んだ時代錯誤的な儒学者であり、また時勢に手遅れの認識もみられるが、それは彼が誰よりもまじめな儒学者であったことに起因するものであり、さらに彼の主張は後日の露骨な儒教的皇道主義とは違うものがあることを論じた。", "keywords": [ "元田永孚", "皇道主義的儒教", "革命論", "血統論", "皇道の訓解", "Motoda Nagazane", "Emperor centralist Confucianism", "theory of revolution", "theory of lineage", "Lessons of the Emperor’s Way" ], "content": "一　はじめに\n\n本稿は、熊本実学派の一員として、人生の後半期に明治天皇の側近となり、当時の大勢の西洋近代化の流れにあって、いわゆる保守反動の儒教主義的な政治・政策の復活を主導した、元田永孚（1818～1891）の活動の思想的意義を論じようとするものである。より具体的には、儒教思想を活動の基盤としながらも、天皇中心の皇道主義をそこに持ち込み、儒教経典を「皇道の訓解」1「皇道の註釈」2 として表現する元田の思想営為の内容如何を問い直してみたい。\n\n沼田哲は彼の『元田永孚と明治国家』の冒頭において、元田を儒学者として規定してよいかという問いを立てて、元田自身の、横井小楠（1809～1869）の実学を承ける儒者であるという言葉を引用しながら、「とりあえずこのように考えておいてよいであろう」と自答している3。元田の儒学者像を改めて強調するような言葉でもあるが、微妙に引っかかる言い方でもある。元田の思想を論じる誰もが認める当たり前のことを敢えて問いかけ、「とりあえず」という暫定的なニュアンスをも与える修飾語によって答えるという、すこし大げさに聞こえるかもしれない理由によるものである。なぜ「とりあえず」なのか、沼田にはこれ以上の説明がないので分からないが、本稿の方向に沿って勝手な想像が許されるならば、元田の思想営為には彼が模範とする小楠に比べて違うものがある、つまり儒教に加えた皇道主義的な言説の標榜がどこかで元田の儒学者像に微妙な影を落としている、ということも関係しているのではなかろうか4。\n\nともあれ、元田の皇道主義的な言説は、しばしば彼の思想営為（儒教思想）を否定的・消極的な方面に向かわせる要素として議論される。主に戦後の評価を指してのことであるが、たとえば、久木幸男は、元田の思想営為を明治儒教思想史の「きわめて特異」なものとして「元田的儒教」と呼び、それがために彼の儒教的な文教政策は「失敗」に終わったと厳しく批判する5。その失敗の主因は、「元田が儒教とは全く異質の「天皇尊崇」を無理に組み入れ、「天皇尊崇」を中心に儒教道徳を再編しようとしたから」6というものである。あるいは、岩井忠熊は、元田を「明治の支配思想としての国家主義思想にもっとも関係の深い儒学者」であるといい、それは元田が天皇中心の政治としての「君権の拡充・絶対化」を目指していたこと、そしてその「君権の制限などということは、思いもおよばぬ」ような「元田の露骨な君主主義は、明治国家の立場において、すでに明白な限界をしめしていた」、と低く評価する7。\n\nもう一つ、これらとは方向を異にして、元田の儒教思想を肯定的に捉える松浦玲の立場も考えてみよう。朱子学゠実学゠政治学としての「儒教型理想主義」という概念をもって、明治 2 年（1869）の小楠亡き後の「儒教型理想主義」の継ぎ手として元田を位置付けるものであるが8、全面的な肯定ではないのが注意を引く。「元田永孚のような、保守反動としての評価が確定している人物をここに持ち込むのは、物騒千万なのだが」9、「晩年の元田に対する是非論はしばらく措くとして」10というような条件付きの評価である。ここでの「保守反動」の意味が西洋近代化に対抗して、その弱点の是正を「孔子の学に求め」るという、儒教復活の主張11に限られるならば、そのレッテルは小楠にも張られるはずであるが、そうではないので、元田のみに適用される別の意味が含まれていよう。そして、それが「元田晩年の是非論」に関わるものとして、彼の晩年に色濃くなる「天祖を敬するの誠心が凝結してその根本をな」すという「国教論」12、さらには「皇室が我が国教育の中心でなければならない」という「国民教育論」13の提唱などを考慮に入れるならば、そのような批判めいた条件付きの評価の背後には、やはり儒教に加えた元田の皇道主義的言説が影響しているといってよいであろう。\n\n以上、すこし強引な引用であったとも自覚しているが、元田の皇道主義的言説が彼の儒教思想への評価を否定的に、あるいは躊躇させる要因になっていることは確認できよう。だが、依然としてはっきりしない問題が残る。前述の引用を中心にしていえば、松浦は、元田が「最晩年」まで朱子学゠実学を堅持したというが14、それならば、その儒教的立場と、評価保留の批判めいた「晩年の是非論」に潜められている皇道主義的立場との間隙はどう埋めればよいのか。\n\nさらにそれは、元田の明治天皇の師傅としての役割においても相反する評価をもたらす。松浦のいう「儒教理想主義」は、具体的に朱子学的原則に従って「君主を聖人にする」ことを前提にするもので15、つまり元田を小楠の「儒教理想主義」の継承者として評価する理由が、彼の「君徳培養」16としての朱子学的帝王学にあった。しかし前に引用した岩井は、元田のその帝王学の任務を「万機親裁」17として「君権の拡充・絶対化」を目指す「露骨な君主主義」といい、評価しない。元田の後半期活動の中心となる帝王学の比重を、松浦は儒教において、岩井は皇道主義において捉えているという、言い換えれば、元田の思想営為（後半期）をめぐって、松浦的な儒教がベースとなる「皇道主義的儒教」なのか、逆に岩井的な皇道が中心となる「儒教的皇道主義」なのかが、衝突しているのである。\n\n元田自身が「夙に尊王経世ノ志アリ」18 と懐述し、実際に儒教に加えた多くの皇道主義的言説が混在している状況において、どちらが優先的で真意であったかといった問いかけは、もはや無理・無意味なものかもしれない。にもかかわらず、こうした問いを立てるのは、少なくとも明治から昭和前期にかけての近代思想史において、元田の「皇道主義的儒教」（あるいは「儒教的皇道主義」）が持つ象徴性が格別だったからである。その時代の思想史が、元田が希望していたものであったか19、挫折・変質されたものであったか20 は別として、近代儒教思想史に与えた元田の影響力が大きかったからである。\n\nたとえば、昭和 9 年（1934）、「日本精神の作興」を求めて創立した「日本儒教宣揚会」が編纂した『日本之儒教』のなかに、当時の大東文化学院教授の藤沢親雄の演説文「明治天皇と元田永孚先生」が収録されている。そこでは、「儒教が我が国に移入せられまして以来次第に我が国固有の皇道即ち天皇道及び国体に醇化致しまして」というこの団体の常套語から始まり、「明治天皇の崇高なる世界観と人生観に対して異帯なる精神的寄与をなしましたのは実に一世の大儒者元田永孚先生其の人であります」と、明維維新の功労者として「大儒者」の元田を持ち上げながら、彼の進講録の一つの「易」の講義を取り上げて、その原理の「王道精神」を「日清日露の戦」や「満州事変」「王道満州国の実現」として捉えている21。\n\nもう一つ、昭和 12 年（1937）、文部省より刊行した『国体の本義』の内容を敷衍解説するために、当時の学習院名誉教授の飯島忠夫に依頼して編纂した『日本の儒教』に載せられている元田に関する言及も、昭和思想界における彼の影響力を窺わせる一つの材料となる。冊子の最尾に置かれている「憲法と教育勅語と儒教」という章のなかのもので、元田を「朱子学者であつて、儒教を以て絶対の真理と信じて居た人である」と紹介し、元田が直間接的に関わった明治憲法と教育勅語について、「推古天皇以来幾多の波乱を重ねて議論されて来たところの日本の道は、この明治天皇が下し賜はつた憲法と教育勅語によつて、明快にして徹底せる解決に達し、従つて儒教の立場もまた明瞭となつた。憲法と教育勅語とは実に皇道の最高経典と謂ふべきである」と述べている22。\n\n元田の儒教に加えた皇道主義的言説は、国民道徳あるいは日本精神といった時代的イデオロギーの創出・普及に欠かせない重要な源泉の一つであった。そうした近代思想史の召喚に応じて、純粋でない保守反動的な儒学者元田のイメージも増幅していったのであろう。この「純粋でない儒教」は、さらにいえば、普遍的儒教と特殊・固有の儒教という問題にも換置できよう。儒教に加えた皇道主義的言説のゆえに「純粋でない儒教」という表現となるが、だからこそ日本の特殊・固有的な儒教ともなる、という意味である。本稿は、こうした問題意識を念頭に置いて、革命論と血統論への認識、「皇道の訓解」の意味という二つに焦点を当てて、元田の儒教に加えた皇道主義的言説の思想史的位置づけを再検討するものである。\n\n\n二　革命論と血統論\n\n前述の『日本の儒教』は、元田（正確には教育勅語）によって「儒教の立場もまた明瞭となつた」といい、その証拠として「儒教思想がその中に混じている不純なる革命思想」を退けたことを挙げているが23、実情はどういうものであったか、元田における儒教的な革命論と皇道主義的な血統論の関係について論じてみよう。\n\n儒教の革命論は、『周易』革卦の「湯武、命を革めて、天に順ひ人に応ず」という語が示すように、民心を代弁する天命に委託して、有徳者による王朝交替を認める論理である。湯武の武力による「放伐」に加えて堯舜禹の間の平和的な「禅譲」を方法的な表象として、革命論は中国と韓国の歴史における王朝交替の正当性を担保してくれるものであった。こうした中国と韓国の歴史に対して、日本思想史では山鹿素行が『中朝事実』のなかで「唯り中国（日本）は⋯⋯天神の皇統竟に違はず」24というように、王朝交替のない「皇統」（天皇の血統）の持続性が強調されてきた。\n\nここで注意したいのは、革命論と血統論は、いずれかの一方に賛成を表明すれば、一方には反対しかない、いわゆる白黒思考のようなものであるかということである。論理的には二項対立的なものであっても、現実でははたしてどうであったか。本稿の目的に沿って先に方向を示せば、革命論（とくに放伐論）に批判的だとして、それがただちに血統論の無条件的な支持にはならない、「中間的な立場」があるということになる。そしてその「中間的な立場」が、君主を有徳者に導こうとする儒教的な帝王学に関わっていることはすでに予測がつくことであろうが、急がずにまずは、二項対立の立場で血統論を支持する側の主張を拾ってみよう。\n\n前述の素行が、中国と韓国の「革命」による王朝交替を「禽獣の相残ふ」25背徳的な行為として非難し、それがなく皇統が続いている日本こそ「中華文明の土」26だと誇っていた根拠は、「中国明かに三綱の遺るべからざることを知る。故に皇統一たび立ちて億万世これに襲つて変ぜず、⋯⋯三綱終に沈淪せず、徳化塗炭に陥らず」27という、儒教的スタンダードの「三綱」にあった。「徳化」を持ち出しているところも、素行の儒学者としての思想的位置を示すものといえよう。\n\nこうした素行式の儒教的スタンダードによる革命論批判・血統論強調の主張は、神道家に至ると、より痛烈な展開を見せる。「禅譲の挙、倫理泯絶の禍を醞醸し、綱常淪斁の災を馴致す。⋯⋯革命の挙、天綱解紐の厄を造し、地維脱結の変を揚ぐ。邪説の魁、暴行の首、孰か焉より大ならん」28と、堯舜の禅譲と湯武の革命（放伐）を区別なく激しく罵る、垂加神道の流れをくむ松岡仲良（1701～1783）の言葉である。王朝交替そのものを全面的に拒否するこうした姿勢の延長線上で、君主の資質などはどうでもよいという、無条件的な血統論支持の主張が登場してくるのは、ある意味当然の流れともいえよう。「只我国ハ昏フテモ天君ハ天君ト仰ギ戴キ奉リ、愚デモ宗領ヲ宗領トタツルヲ比莽呂岐ノ道トス」29 （傍点は筆者、以下同じ）と。\n\nところで、非難の語調や方向は異なるが、神（天照皇）の子孫による統治という日本の特殊性を前提にして、無条件的な血統論を当然視する儒教側の主張もある。元田が自藩の先輩学者を除いて、ほとんど唯一その学問を是認していた熊沢蕃山（1619～1691）の言説がそれである。蕃山は、湯武の徳が堯舜や文王に「及ばずして」、「放伐」という「徳に恥る」ことをやってしまったと30、儒教の放伐論に冷ややかな態度を取りながら、日本の永続的な天皇統治の歴史を「必然の理」として、次のように擁護する。\n\n天照皇は地生にをはしまさず。神武帝、其御子孫にして天統をつぎ給へり。⋯⋯然れども一度たゞ人となりぬれば、天統をつがず地生にひとしきゆへに、天下をとりても帝王の号を得事不叶、三種の神器を身にそへ奉りて、天津ひつぎをふまん事は、天照皇の恐多く、且天威のゆるさぬ所あり。日本のあらんかぎりはかくのごとくなるべし。他の国はなき例なれ共、日本にては必然の理也31。\n\n天皇統治の当為性を、他国には例を見ない日本の固有の現象として打ちだす蕃山の主張は、彼の儒教受容の方法としての「時処位」論に基づくものであろうが、その天皇の資格を「たゞ人」の「地生」ではないところに求めていることは、やはり注意を引く。「天照皇」の血統は天皇統治を権威づける条件となるが、一方では「天統」「天威」のみにすがることによって、人君に必要な統治資質の涵養を疎かにし、または現実政治での役割を制限する論理にも繋がっていくからである。次の蕃山の言葉を見よう。\n\n代をかさねて天下をたもつは天の廃する所なりといへり。しかれ共、王者は天神の御子孫にして地生にあらず。ことに日本にをいて広大の功徳をはします故、天下の権勢をばさり給ひて、やはらかにして上におはしませば、いつまでも日本の主にてをはします道理にて侍り32。\n\nここで蕃山は、血統論を問題視する『孟子』の論理を引き合いに、日本の「天神の御子孫」による世襲的統治を肯定している。だが、その天皇の「いつまでも日本の主にて」という永続的な地位は、「天下の権勢をばさり給ひて、やはらかにして上におはしま」すという、現実政治に関与しないことを条件としたものであった。徳川政権の下での自己検閲的な発言といえばそこまでであるが、蕃山には後醍醐天皇のような「王徳」のない天皇の政治参与を良しとしない言葉も見える33。長い戦乱の末にやっと安定を取り戻した当時の秩序が、「天皇、道をしろしめさず、賢良を用ひ給はず、昔と時勢のかはりたる事をしり給はざりし」34というような存在によって、再び乱されることへの不安も、蕃山の天皇の政治参与を抑制する主張の一因であったに違いなかろう。\n\n天下の人是を見て、威も力もなき人を日本の主筋とし、かくのごとくあがめ奉り主君となしてかしこまり給へるは誠に道ある君なり、我等いかで国・郡を給はりながら忠を存ぜざらむやと、むかし賊心ありし者も、たちまちひるがへして譜代の思ひをなせり。こゝを以世の太平すみやかなり。禁中をはしまさではいかで此徳あらんや35。\n\n「天下の人是を見て」の「是」はこの文のすぐ前において、戦国時代が収まり、武家の将軍と諸大名が参内して宮中の礼儀・音楽を見聞して感動するという光景をいう。「天下の人」が、武将たちの「威も力もなき人を日本の主筋」として崇敬する様子を見て、自分たちもそれを模倣することで「世の太平」が実現されるようになった、と蕃山はいう。要するに、「天皇の存在」そのものが、天皇の価値であり存在理由であるというものである。蕃山は、天皇に現実政治を主導していく役割を求めず、当然そこでは「王者」になるための積極的な帝王学も必要ないことになる。この点、元田とは大きな開きがある36。\n\n元田は、蕃山が経験していない天皇の「侍講」として、彼自身の表現で「未タ聖知発達ノ機ニ至ラス」37、「全体遅鈍の御天資に而、一と通り奉接候ては乍恐御不分りの様に奉伺候処」38、「或は狭急偏執の御失無きとも云難きを以て」39などといった、「地生」的な側面に対面していた。そこで、元田は「君徳培養」の教育を強調し、その教育は「人君」たる存在の必修要件であると、次のように進言する。明治天皇への初進講のときの言葉である。\n\n抑人君天資ノ聡明学フニ頼ルニ非サルカ如シト雖トモ、苟シクモ智ヲ恃ンテ自ラ用ヰル時ハ其知ル所狭小ニシテ過不及ノ誤リアルヲ免レス。是ヲ以テ聖帝明王ハ必好ンテ聖人ヲ師トシ、其則リヲ取ル40。\n\n「人君」は血統的な資質に頼るのではなく、「聖人ヲ師トシ」て教育を受けなければならない。「聖人」は具体的に孔子、広くは儒教の教えをいう。そして、元田は、もし「人君」が教育を怠って「不徳」と見られる場合には、「環て視る者幾千万、其知識の日新、事業の月盛、我を蔑如する何を憚て為ざらんや」41と、人々から蔑視される事態を招くことになると諫める。さらには、「人君にして一念愆れば四海億兆の憂となり、一日怠れば千百年の患を貽す」42といい、「人君」の教育は国家の「治乱興亡」に関わる大事な問題であると進言する。前述の松岡仲良の「昏フテモ天君」論はいうまでもなく、蕃山の「天統」「天威」に満足する血統論とも、異なる方向である。元田は単なる血統論の支持者ではないのである。\n\nしかしとはいえ、元田をその反対の革命論の支持者として想定することも無理な話である。いうまでもなく、「大日本国は天孫一系の皇統万世に君臨す」43「皇統一系天壌窮り無し」44「神胤一系万古不易」45といった血統の永遠性をいう主張も、彼の思想活動の重要な一側面であったからである。もし彼のこのような言葉を、天皇の側近という地位による、あるいは歴史に再び呼び出された王政復古という時代的特殊性を反映する、たとえば徳富蘇峰が評した「高尚なる臨機応変者」46としての立場をあらわすものだとすれば、次の言葉には儒学者の一般論として革命論に反対する彼の立場が窺える。明治 16 年の新年進講の際に『論語』「道之以政」篇を講義するなかで持ち出した言葉である。\n\n彼国テ堯舜禹ノ三代ト周公ノ成王ヲ輔ケテ天下ヲ治メマシタル世ノ三、⋯⋯其余ハ多ク天下ヲ奪ヒ取リマシタル故ニ、所謂利己主義ニテ、初メヨリ人君ノ徳ヲ以テ天下ヲ治メマスルノ主義ニテハコサリマセス。湯武ヲ始メマシテ斉桓・晋文・漢ノ高祖・唐ノ太宗・宋ノ太祖・明ノ太祖等孰レモ、己レ天下ヲ取リテ我功業ヲ為サント欲スルノ利己主義ヲ免レマセヌ故ニ⋯⋯47。\n\n元田は、中国の王朝交替を「利己主義」によって「天下ヲ奪ヒ取」る行為だと指摘し、湯武の放伐をその利己主義の元祖的な行為として、蕃山よりも強い語調で批判していた。元田は革命論に反対しているのである。\n\nこのように論を進めてくると、元田は革命論と血統論をめぐって自己矛盾に陥っているかのように見えるが、しかし思想史における儒学者一般の見解はどうであったか、と問いを広げてみれば、別の視点も得られよう。儒学史において革命論に積極的な賛成を表した学者は、いったいどれほどであったかという問いである。\n\n近代以前の学者のなかで革命論を支持した学者として有名なのは、明清の王朝交替期を経験し満州異民族の支配に絶望した黃宗羲（1610～1695）くらいであろう。彼は『明夷待訪録』のなかで、「小儒は規規焉として、君臣の義を以て天地の間に逃るる所無しとす。傑・紂の暴に至りても、猶お湯・武当にこれを誅すべからずと謂いて、妄りに伯夷・叔斉の無稽の事を伝う。⋯⋯後世の君、父の如く天の如きの空名を以て、人の窺伺を禁ぜんと欲する者は、皆なその言を不便とし、孟子を廃して立てざるに至る。源を小儒に導くに非らずや」48、と革命論支持の論陣を張った。「小儒」たちの間違った世論操作によって、湯武の放伐よりも伯夷叔斉の荒唐無稽な故事が宣伝され、遂に孟子の革命論が廃止されるに至ったという論弁である。「天下の大害を為す者は君のみ」49という当時の君主制度を追っ払うためには孟子の革命論の導入が必要だという主張であるが、この言葉はかえって革命論が現実の思想界においては支持を得ていない寂しいものであったことを浮き彫りにしている。\n\n孟子の革命論を公開的に攻撃した代表的な言説の一つに、北宋の名分論者の司馬光（1019～1086）の主張がある。司馬光は、孟子が斉の宣王を相手に「君に大過有れば則ち諫む。これを反覆して聴かざれば則ち位を易う」50と言ったことに対して、「孟子の言、以て驕君の非を格すに足らずして、ただ以て纂乱の資を為すに足るのみなり」51と非難していた。\n\nところが、司馬光が指摘する「簒乱の資」は、実は孟子も心配していたことであった。「世を継いで天下を有つ、天の廃する所は必ず傑・紂が若き者なり。故に益・伊尹・周公、天下を有たず」52と。すでに引用したように、蕃山はこの言葉を「代をかさねて天下をたもつは天の廃する所なりといへり」といい、孟子は王位の世襲そのものを否定しているかのように解釈しているが、この言葉の核心は「必ず傑紂」というところにあろう。孟子は、「天の廃する」放伐の対象に厳格な制限を掛けて、「簒乱」を禁止し、世襲も認めていたのである。さらに、「世衰え道微にして、邪說暴行有た作る。臣にしてその君を弑することこれ有り、子にしてその父を弑することこれ有り。孔子懼れて春秋を作る」53として、臣の忠的な論理を強調する『春秋』の大義名分論を宣揚したのも、孟子にほかならなかった。\n\nそして、朱子は、孟子の革命論をあらわす、あの有名な「斉宣王問湯放桀」章の注釈において王勉の言葉を借りて、「ここの言は、惟だ下に在る者に湯武の仁有りて、上に在る者に桀紂の暴有るのみは則ち可なり。然らざればこれ未だ簒弑の罪を免れざるなり」54と、放伐論に制約をかけることを忘れない。こうしてみると、孟子に代表される儒教の革命（放伐）論は、君主を誡める意味が強く、実際の政権交替を扇動するものではなかったといえよう55。言い換えれば、革命論は専制君主の悪政を防ぐための一種の警戒装置として、その実行を要求する儒学者は、少なくとも平時においては希少であり、大勢は血統による世襲を認めていたということである。\n\n平時においては王位の世襲が当然のように受け入れられているなかで、儒学者たちが理想とする王道政治を目指して君主の有徳を確保しようとした制度が、まさに「経筵」であり「侍講」制度であった。朱子が 65 歳にして煥章閣代制兼侍講に任命されて即位したばかりの寧宗に『大学』を進講したことや、朝鮮の李退渓（1501～1570）が 68 歳にして大提学・知経筵の任務で 16 歳に王位に就いた宣祖を相手として「戊辰六条疏」を上書し、「四箴」、「西銘」などを進講し、また「聖学十図」を献上したことは、その一例といえよう。元田が革命論を支持せず、かつ血統論に一定の距離を置いて「君徳培養」の教育を強調してやまなかったのは、それこそ儒教の伝統に倣って忠実に再現しようとしたことであった56。\n\nしかしそれにしても、今一つ釈然としない問題が残る。元田がそのような儒学者一般の立場を飛び越えて、「天孫一系」の日本的血統主義の主張を人一倍強く言い出していったのは、どのように理解すればいいのかという問題である。やはり皇道主義者の面貌によるものであるか。それとも、前述した徳富蘇峰の「臨機応変者」や小楠が批評する「利口者」57としての俊敏な状況認識が働いているものであろうか。要因は一つというより、濃淡の割合はともかくとして、幾つかの因子が複合的に絡んでいるのであろう。ならば、彼の儒学者としての立場により密着して、日本的血統主義の主張においてもそれは貫徹されているのか、もしそうだとすればその原理は何であったかを探してみることも、無意味ではないだろう。「華盛頓」論がその一端を窺う手がかりとなる。\n\nアメリカの初代大統領のワシントンを、小楠が堯舜のように高く評価していたことはよく知られているところであるが58、元田もその見解に同意し、むしろ小楠にはない、「華盛頓賛」「贅言」という専門的な一文を書くほどであった59。「華盛頓賛」では、アメリカ独立戦争におけるワシントンの姿勢を、周の武王が殷の紂を討つ際に発したという『書経』泰西篇の内容に比肩し、さらにワシントンが大統領に就いて 8 年間務めた後に「位を辞し職を譲り恬然として旧閭に帰」ったことを、堯舜の「禅譲」と同様に捉えて、「其の聖徳大業豈堯舜と道を同じうする者に非ずや」と述べていた60。「贅言」は、そのようにワシントンを称賛することは、西洋の「共和政治」を優越的なものに考えて、日本の「皇国の国体」を無視し貶めることではないか、という周囲からの批難に対して、それを弁解する内容のものである。\n\n我が先皇の国を建つる、天孫の始めて茲の土に降臨したまひしより既に四海を以て一家と為したまひ、万民を視たまふこと赤子の如くその惻怛愛国の至、親ら其の労に任じて他人に託するに忍びたまはず、故に皇統一系万世に伝へて易りたまはず、其の恩沢寰宇に弥満し骨髄に浹洽す。⋯⋯彼の四年にて交代し民に常主無く自由に政を為すものと其の体裁固より同じからずと雖も、然れども其の天下人民の為にして一毫己が為にせざるの心は未だ曽て一ならずんばあらず61。\n\n「皇統一系万世に伝へて易りたまはず」と、日本的血統主義を擁護する姿勢は見てのとおりである。ここで注目したいのは、そうした「皇統一系」の「国体」は、「四海一家」「万民赤子」「天下人民の為にして一毫己が為にせざる」といった、いうまでもなく儒教の「王道政治」的な論理によって支えられてきた、という元田の言い分である。それは歴史的な事実というより、今侍講を務めている儒学者元田の責任意識・政治的目標が投影されているものと見るべきであると思うが、それはともかく、元田は続いて次のように天皇の世襲政治とワシントン的な共和政治の内実を、儒教の王道政治において一致させている。「嗚呼君臣の礼正しくして愛敬の道明らかに、同胞の義篤くして共和の実行はれ、法令簡明にして強制無く、民をして分に安んじ業を楽しみ各自主の域に至らしむるは是我が邦古来の国体なり。今果して能く是の如くんば則ち華盛頓も亦将に甘心せんとす。夫の君威を主とし束縛を専らにするが如きは則ち覇政の陋習我が皇国の体にあらざるなり」62と。「皇国の体」は「覇政」ではない「王道」を本質とし、その点においてワシントンも日本の血統主義に「甘心」するだろう、というのである。\n\n「此一句ハ舜禹授禅ノ要旨乃伝国ノ要言、我朝ノ三種ノ神器ノ意義ノ如シ」63とあるのは、元田が明治 18 年（1885）の書経講義のなかで堯舜禹の間の「禅譲」の際に発せられたという「人心惟危～允執厥中」句を解説したときの言葉である。儒教革命論の一軸をなす「禅譲（授禅）」を拒否するところか、日本的世襲論の象徴である「三種ノ神器」と同格の事柄として譬えている。前記した神道家の松岡仲良からすれば、到底あり得ないことになろう。といっても、これは元田が内心では革命論に加担していたなとではなく、革命論か血統論かの二項対立的な思考を乗り越えて、というより、彼の関心はそういう理屈的なところにはなく、堯舜禹と古代日本に共通して行われていたという理想社会を、この時代にもう一度実現したいという願望の表出として捉えるのが妥当であろう。共和制のワシントン賞賛もそうした脈略で理解すべきであろう。\n\nだが、元田の「華盛頓ハ堯舜以来ノ聖人、或ハ優ル所アルモ知ルヘカラス」64といった評価は、出仕後の後半期に至ってはほとんど見られなくなる65。しかし、ワシントン論に投影されている儒教の理想社会を実現すべく、その中心となる君主に対する教育の重要性を強調する姿は、晩年まで変わることがない。次の言葉は元田 70 歳のとき（明治 20 年）の新年進講で『周易』の乾卦を講義したもので、「君体」として血統的な世襲を前提し、「君徳」培養を力説している。\n\n御国ノ御君体ハ、全ク天道易理ニ御符合ノ御正体ニテコサリマスル、其君体ハ天ノ変リナキカ如ク一定不易ノ者ニテコサリマスレトモ、君徳ニ於キマシテハ、乾々剛健ニシテ息ミマセス、須臾モ撓ミ怠リナク、朝廷百官天下万民ニ先立チテ、活溌運動天下ヲ率キ回ラス程ノ誠心カ、大土台ト相成リマシテ⋯⋯66。\n\n「御国ノ御君体」つまり日本の皇統が間断なく継続しているのは、「天ノ変リナキカ如ク」当然のことであるが、それでも「君徳」は「須臾モ撓ミ怠リナク」錬磨しなければならない。さらに、元田の講義は次のように続く。\n\n支那ヲ始メ外国ノ君体モ御国ト同ヤウニ万世不易ナル筈ニテコサリマスルニ、君体ノ動キナキニ泥ミマシテ、君徳モ怠リ滞リマシテ進ミマセヌヨリ、終ニ伏犠以後革命ノ君トハナリ降リマシテコサリマスル。御国モ中世以後ハ恐レナカラ君体ノ動キナキニ御安心ニテ、君徳ノ剛健ヲ御欠アラセラレマシタル故、適々御衰微ニモ相成リコサリマスルカ⋯⋯67。\n\n中国の「君体」も日本と同じく「万世不易ナル筈」というのは、元田が王朝の世襲を日本だけの特殊事情ではなく、当たり前の一般論として考えていたことを伝えていよう。元田はそのうえで、中国では革命にあって王朝交替が繰り返され、日本では革命はなかったものの、王朝が「衰微」に陥った原因は、「君体」の不変性に安心して「君徳」培養に怠慢であったことによると指摘している。血統論を象徴する「君体」は既定の事実として、元田の主な関心はもっぱら「君徳」に注がれていたといえよう。だからこそ、「人君⋯⋯万一誠敬ノ心弛ミマシテ、一身ノ私欲ニ流レ怠リ荒ミマスレハ、天命忽チニ人君ノ身ヲ離レマシテ、天下ヲ保タレマスルコト相成リマセス」68という、より革命論の雰囲気を色濃く漂う、いわゆる「天命無常論」を明治天皇の前で進講しえたのであろう。\n\n要するに、革命論を是認せず血統論を既定の事実として、なおかつ王道政治実現の主体たる時の君主の君徳培養に力を傾注しようとしたのが、この問題における元田の変わらない姿勢であった。それは東アジア思想史における、とくに日本においては蕃山をも乗り越えての69、典型的な儒学者の立場を示すものであったといえよう。次に節を代えて、この元田の儒学者としての立場は、儒教経典を「皇道の訓解」などと位置付けるところでは、どのように表明されているのかを考えてみよう。\n\n\n三　「皇道の訓解」\n\n前で元田が儒教の「禅譲」を、「三種の神器」の伝達の意味と同じく捉える言葉を引用したが、神道（皇道）の意味を儒教経典によって裏付けようとする具体的な取組は、この「三種の神器」をめぐる言及から確認することができる。次は、元田が侍講として出仕する前年の明治 3 年（1870）に記したもので、ここでは儒教経典を「神教の注解」といっている。すこし長文になるが、煩をいとわず引用してみる。\n\n①蓋上古の神聖聡明叡智の性、これに加るに寛容温厚発強剛毅の徳を以て天下に照臨し、教を万世に垂る。此時に当り文字の記するなく、書契の伝るなし、即ち地に対し日用に象なく、教を三種の神器に寓して、天下万世をして見て覚り易からしむ。蓋鏡の象は明なり、所謂聡明叡智の性なり。玉の象は仁なり、所謂寛容温厚の徳なり。剣の象は義なり勇なり、所謂発強剛毅の徳なり。②此三徳民生の始、之を天に受け性とする所のものは、人々固有せざることなし。故に人君能敬して之を存し、推して以て天下に及せば、天下の人亦此三徳を明かにして、天下国家治平ならざることなし。③教約して旨遠し。実に言語文字の及ぶ処にあらず。神武天皇尊崇体認、崇神天皇欽承継述、応神天皇に至り始て西土の経典を加えて神教の注解となし、益斯道の精義奥旨を推演して、学路用功始て明かに、聖徳広運人に取て養を為すの誠意、実に堯舜と道を同ふす70。\n\n番号をつけて内容を三分したのは、今後の論議進行のために、いわば総論のつもりで設けたもので、それぞれこの問題に臨む元田の認識の方向が示されていると思ったからである。①では、「三種の神器」が象徴している徳目を、『中庸』の「智仁勇」の三逹徳にまっすぐに配当している71。最初の「蓋上古の神聖聡明叡智の性、これに加るに寛容温厚発強剛毅」という天照大神の徳を描写する語句は、『中庸章句』 31 章の言葉を模倣したものである72。②では、神器の三徳を、天から人々に賦与されている「固有の性」、つまり『中庸』首章の朱子注釈に基づいて生まれながらにしてすべての人々に内在する人間の本性・本質として提示している。三徳の所有を天皇に限らず、衆人に広げて一般化・普遍化するという、ある意味大胆な解釈となるが、その拠り所もやはり朱子学である。そしてその観念において人君（天皇）が先頭に立ち、三徳としての人間の本性を明らかにしていく、という統治原理が示されている。③では、「教約して旨遠し」の神器の真意が、応神天皇の『論語』の受容・伝達によって「神教の注解」となり、儒教と「道を同ふす」という、いわゆる神（皇道）儒一致論が主張されている。一つずつ、もうすこし詳しく検討してみよう。\n\nまず①での、元田が「三種の神器」の徳を『中庸』の「智仁勇」に配当して解釈する立場である。元田の解釈は、日本思想史の伝統を背景にしたものであるが、実はその伝統は元田の立場ほど明確ではない、というところに注意したい。元田による両者の結びつけを「まっすぐに」と表現したのは、それを念頭に置いたものである。元田が神器の意味をめぐって日本思想史のなかでとくに言及しているのは、北畠親房と熊沢蕃山である。\n\n其後源親房神皇正統記ヲ著ハシ、三種神器ノ徳ヲ説キマスルニ、此中庸ヲ引証ニ致シテコサリマシ、熊沢了介ニモ三種神器ノ註解ハ中庸ニシクハナシト申シテコサリマスレハ73、\n\nところが、親房の『神皇正統記』の解釈は、元田の言明とは違って実際には「中庸ヲ引証」していない。『神皇正統記』には、「鏡ハ一物ヲタクハヘズ。私ノ心ナクシテ、万象ヲテラスニ是非善悪ノスガタアラハレズト云コトナシ。其スガタニシタガヒテ感応スルヲ徳トス。コレ正直ノ本源ナリ。玉ハ柔和善順ヲ徳トス。慈悲ノ本源也。剣ハ剛利決断ヲ徳トス。知恵ノ本源也」74とあり、智仁勇ではなく、「正直」（鏡）、「慈悲」（玉）、「智恵」（剣）を表象している。『中庸』の内容とは名目も異なり、徳目の配当もずれているのである75。和辻哲郎が、「親房のこの解釈は極めて明瞭であつて、儒家の智仁勇三徳による解釈と混同される筈はないと思はれるのであるが、どういふ理由によつてか、少なからぬ学者がこの混同に陥つてゐる」76と指摘したとおりである。\n\nもう一つ、元田は、蕃山の神器論も自説の典拠として引用している。蕃山の『大学或問』には、「智・仁・勇は天下の達徳なり。此三種の象を注解して経伝とせば、これに過たる神書あらじ。三種の注解は中庸にしくはなし」77という言葉がある。しかし蕃山の神器の解釈は、『中庸』のみの忠実な反映ではないのが、元田の立場と比較される。蕃山は『神道大義』のなかで「夫神道は正直を以て体とし、愛敬を以て心とし、無事を以て行とす」78と「神道」を定義しながら、それらの徳目は儒教においては「智仁勇の三にかよへり。正直は知なり、愛敬は仁なり、無事は勇なり」79と対応させる。また『三輪物語』では、「鏡は心の神明にして、虚霊不昧にかたどれり。天にありては日光とし、事におきては正直とす。玉は心の温潤にして、慈愛恭敬なるにかたどれり。天にありては月光とし、事におきては委曲とす。剣は心の剛強にして、堪忍裁断なるにかたどれり。天にありては星光とし、事におきては威武とす」80と解釈する。\n\nこうした蕃山の解釈について、和辻哲郎は、「彼もまたこれを智仁勇の「象」と解するのではあるが、しかし正直、慈愛、断の三者をそこに結びつけざるを得なかつた」といい、「ここに太神宮の神託が影響してゐることは否定できないであろう」と指摘している81。蕃山の解釈には依然として日本固有の神道的な立場が残っているという主張である。つまり、その蕃山の解釈との比較において、神器゠天照大神の徳を『中庸』の言葉に即してそのまま忠実に引用する元田の、儒教中心の解釈が浮き彫りになってくるのである。ちなみに、儒家神道を主張した山崎闇斎は、「鏡は分明なるを表し、玉は曲妙なる心を表し、剣は知恵の利を表し」82と、神道的な解釈を示していた。こうしてみれば、儒教理論を利用して神道を説明するという神儒一致の儒家神道は、明治の元田に至って「粹然たる」理論的な一致をみた、といっていいかもしれない83。\n\n次に②での、神器の徳目を人々の普遍的な性とし、それを統治原理に結び付ける見解を考えてみよう。まず普遍的な性に関して、すでにそれは朱子学の影響によるものといったが、具体的には、元田の「之を天に受け性とする所のものは、人々固有せざることなし」という言葉が、『中庸章句』 1 章の「天命之謂性～修道之謂敎」に対する、「蓋し人、己の性有るを知りて、其の天に出るを知らず、⋯⋯聖人の教有るを知りて、吾の固有する所の者に因りて、之を裁するを知らず」84という朱子の注釈に基づいていることによる。神器の徳目を人間の本性として把捉する元田の別の言葉を紹介すれば、「天祖ノ詔ハ乃此処ノ天命スルノコトニテ、三種ノ神器ハ乃此処ノ之ヲ性ト云コトニテコサリマスル」85、「故に上古の神聖三種の訓を示し、人々本性の徳を覚りて、之を全ふせんことを欲するの皇極とし、其これを学ぶの道は堯舜禹の欽明精一執中、孔子の克己復礼、大学の格物致知誠意正心修身、中庸の博学審問慎思明弁篤行、孟子の察識拡充一として本性に復るの法に非ざるはなし」86、などがある。\n\nところで、「天命の性」としての人間の本性は聖人と衆人との間に区別がない、という朱子学の議論は、孟子の性善説を引き継いだものである。朱子は、「聖人も我と類を同じくする者なり」という『孟子』の言葉について、「聖人も亦た人ならくのみ。其の性の善、同じからざる無きなり」という注釈を施した87。だが、聖人と衆人が同じであるというのは、あくまでも善なる本性という抽象的・理論的な領域においてのことで、現実的な身分や階層の差異を無視する、いわば近代的な意味での人間平等論的な主張ではないことはいうまでもない。「聖人も人間であるにすぎない」という朱子の注釈には、聖人の特別な地位を奪うのではなく、誰でも聖人になりうるという衆人側の道徳的な奮発を催促する意図が込められていよう。朱子学では聖人に到達するための具体的な方法を「居敬」と「窮理」として定型化したが、元田のいう、これまた朱子学の重要な理論の一つである「復性論」に触れて「本性に復るの法」として列挙している堯舜禹から孟子までの工夫法が、それを反映していることは疑いない。神器が表象する徳目の所有を天皇に限定せず、人々の普遍的な性として位置付ける元田の解釈は、そうした朱子学の立論を駆使して立てられたものであった88。\n\nそして、元田はその朱子学的本性論を彼の構想する統治原理に適用していくが、この論法は明治 17 年（1884）、伊藤博文に提出した「国教論」89という一文にも一貫してあらわれているので、それを引用して元田の立場を検討してみよう。\n\n蓋し我の所謂天祖を奉ずるとは、かの釈迦を奉じ耶蘇を奉ずるその道固より異る。而して又祠官誦呪の如きに非ざるなり。天祖の徳は智仁勇にして人心の神府に賦在す。特に天祖の独得にあらずして伝へて列祖今上の神髄にあり。特に列祖今上の所有ならずして、人々の固有するところなり。故に吾が霊智を磨き、吾が大仁を拡げ、吾が神勇を振ひ、以て身を修め、国を治め、天下を平かにするは、乃ち我が天祖を奉ずる所以なり90。\n\n「天祖の徳」゠智仁勇が天皇のみならず人々にも内在しているという主張が、ここにも繰り返されているのは見てのとおりである。元田の持論の一つといっていいだろうが、それはともかく、ここで注意したいのは、その主張が向かう帰着地である。元田は、「人々の固有するところ」となった「天祖の徳」を、今度は各自の努力で極大化して修身治国平天下に貢献する、それが「我が天祖を奉ずる所以」であると論を展開していく。つまり、朱子学の本性論を借りて三種の神器を説明する目的が、「天祖を奉ずる」として皇道を絶対化するところに収斂されていくのである。別の言葉を引けば、「今日ノ急務ハ天祖ノ詔ニ基ツキ神器ノ徳性智仁勇ノ三徳ヲ拡張致シマシ、忠孝ノ教ヲ修メマシテ」91というものである。\n\nしかし、元田の「天祖を奉ずる」ということは、人々に求められる一方的な責務ではなく、ある先決条件の下で出されたものであることを見逃すべきではない。君主（天皇）の先導と百官の補佐を、人々の責務に先立って要求しているということである。上引用に続く言葉の、「故に天子これを用ひ、以て明徳を天下に明らかにし、大臣百官これを用ひ、以て天子を輔けて国家を治む、士民これを用ひ、以てその身を修め、その所を安んず」92という、天子―大臣百官―士民の順番で、それぞれが担う違う役割を挙げていることがそれを示している。さらに、「国教論」の別バージョンでは、はっきりと天皇以下の政治指導者たちの「率先」を以下のように強調している。\n\n其の天祖を奉ずるや、非礼拝祭祀虚文の謂に非ざるなり。即ち天祖の教を奉ずるなり。⋯⋯今天子と皇族大臣群僚百辟、一意にして之を奉じ、確乎として抜かず、以て億兆に率先するときは、則ち億兆亦た将に感じて興起することを観る所有り93。\n\n「億兆」の責務に先立って「天子」の「率先」を要求するという観念は、すでにこの節の冒頭で引いた明治 3 年の「教育大義私議」のなかの「人君能敬して之を存し、推して以て天下に及せば」という言葉にも示されているが、それが『大学章句』経 1 章の「大学の道は、明徳を明らかにするに在り、民を親たにするに在り」という、これまた朱子学的な統治原理に依拠していることも容易に推察することができる。\n\nもう一つ引けば、『論語』為政の「道之以政」章の講義において、「徳ハ即チ人君躬ニ行ヒ心ニ得ル所ノ実徳、譬ヘハ人君躬親ラ孝弟ノ実ヲ尽シテ民ヲ孝弟ニ導キ、仁義ノ実ヲ尽シテ民ヲ仁義ニ導キ、躬ニ節倹ヲ行フテ民ヲ節倹ニ導キ、躬ニ天職ヲ尽シテ民ヲ職業ニ導クト云カ如ク、未タ人民ニ法令ヲ下スヲ待タスシテ、人君躬親ラ其行フヘキノ道ヲ尽スヲ此徳ヲ以テスト云コトナリ」94と、道徳の実践から日常の節約・仕事に至るまで人民に先立って「人君躬ニ行ヒ心ニ得ル」ことを要求する言葉からも確認することができる95。\n\n要するに、元田が「国教」として天皇を神聖視し皇道を絶対化するといっても、それは無条件的な追従ではなく、無制限の君権の拡張を助長するものでもなく、君主（天皇）が「率先」して「躬ニ行」う、という儒教政治理論の伝統的教義をベースに置くものであった。こうした主張の延長線上で、③の「神教の注解」としての、儒教と「道を同ふす」という神儒一致論が出されてくるのであるが、しかしそこには全く一致とは言えない事情も見え隠れしている。次にそれを検討しよう。\n\n元田が『論語』やそのほかの儒教テキストを「神教の注解」「皇道の註釈」あるいは「皇道の訓解」などとして位置付ける意味を、彼の言葉で探してみれば、「孔子ノ道ハ書ニ伝ヘ、我先王ノ道ハ神器ニ寓ス、書ニ伝フ故ニ講誦シテ教ヘ易ク、器ニ寓ス故ニ深奥ニシテ識リ難シ、唯道ハ一ナリ、故ニ書ヲ以テ器ニ参ヘテ其義覩ル可シ」96、「夫道は太神の訓に存す、人々自求めて足れり、然れども存誠致知の方、克己復礼の目、天下経綸の道、聖経に由らざれば其詳なることを得ず」97、などがある。神教・皇道と儒教の道は内実において同じであるが、神教の内容は奥深くて知り難いものなので、礼儀・道徳論から政治論に至るまで具体的な方法を具えて分かりやすく教えている儒教思想によって補うことをいうものである。しかもその儒教の受け入れは、選択的な補完ではなく、「書ヲ以テ器ニ参ヘテ其義覩ル可シ」「聖経に由らざれば其詳なることを得ず」というように必修的な補完として説かれている。いかにも儒学者としての元田の面貌を覗かせているものといえるが、それと関連して注意したいのが、応神天皇の儒教導入の宣揚と『論語』の特別な位置づけである。\n\n唯此論語ノ書孔子ノ道ニ於テハ支那ノ書ニシテ我朝ノ伝書ナリ。孔子ノ道ニシテ我日本人ノ道ナリ。⋯⋯扨又此書ハ辱クモ応神天皇ノ御伝書ナリト尊信スベキナリ。⋯⋯此論語ハ即チ天皇伝授ノ御書ト云テモ可ナリト存シ奉ルナリ98。\n\n『論語』は中国の書ではあるが、応神天皇の「伝授」によって「我朝ノ伝書」となったという。これに加えて、\n\nこの書は是れ応神帝伝授の書にして、皇道の訓解なり。何を以て之を云ふ、蓋し我朝にて道学を講ぜしは、帝より始まりて、我朝の書、此の書を以て訓謨の権輿とす99。\n\nという言葉をみれば、元田において「皇道の訓解」と「我朝ノ伝書」は置換可能な概念であったといえる。重要なのは、元田が応神天皇による『論語』の「伝授」「伝書」を、文字や儒教の興起といった単なる先進文化の導入を超えて、「日本人ノ道」、または日本という国家の大計のはじまりをなす「訓謨の権輿」として、より日本的な特殊性を持たせながら位置付けている点である。この捉え方は、先輩学者たちの応神天皇の『論語』導入の意義を語る、たとえば、北畠親房の「此国ニ経史及文字ヲモチヰルコトハ、コレヨリハジマレリゾ」100、山崎闇斎の「是日本経学のはじめなり」101、山鹿素行の「ここに於て始めて書籍を伝へ、大いに儒風を闡き、文教の興ること誠にここに在り」102といった一般的な文化現象の興起を伝えることと比較すれば、その特別な位置づけが鮮明になる。それは言ってみれば、伊藤仁斎が『論語』を「最上至極宇宙第一の書」103として、「万世道学の規矩準則なり。⋯⋯万世に通じて変ぜず、四海に準じて違わず」104という普遍的真理の側面を評価したとすれば、元田は「日本人の道」として日本の特殊・固有の側面を『論語』に託してあらわしていたともいえよう。\n\nそして、元田が固有の「日本人ノ道」として特に強調したのが「忠孝」であるが、この徳目の解釈においては本来の儒教との衝突が生じている。次は明治 11 年（1878）の経筵での「論語弟子入則孝章」講義の一節である。\n\n我が国は天地開闢より天祖の一君ましまして、臣民を統治し子々孫々万世窮りなし。故に天下の大道は君臣に始まりて、万づの道理皆此の君臣に包含せり。⋯⋯君臣の忠義と父子の親愛とを合一にしたる、世界無比の至道純理なれば、堯舜も夢にも見ず、孔子もいまだ説き出すこと能はざる所なり。此の君臣の大道、上下古今に貫通し、其の中に父子の親、夫婦の和、兄弟の序、朋友の信、其の余細大の道、悉く成り立つなり105。\n\n元田は、「忠」を君臣間の徳目として解しながら、日本では歴史的にその「忠」と父子関係の「孝」（親愛となっているが、この講義の主題や脈絡からして孝にいい直して差し支えなかろう）との「合一」がなされてきたという。そして、その二つの徳目を「合一」的に捉えることは、堯舜や孔子もできなかった、日本のみに伝わる「世界無比」の特殊の観点であり、さらに君臣間の「忠」はあらゆる道徳を包括する一番上位の徳目である、と説いている。続く言葉では、「忠義を重んじ、三歳の童子も忠孝に死することを忘れず。我が国固有の道にて」106といい、忠孝の日本的な固有性を強調している。\n\n元田は以上のことを『論語』の孝弟概念に即して示しているが、儒教思想史の脈絡と齟齬しているのはいうまでもない。元田は忠の徳目を君臣関係において見出しているが、その原義はむしろ、『論語』の「人の為に謀りて忠ならざるか」107などの自分の誠意を尽くすという対自徳目として理解するのが一般的である。また、忠と孝を合一的に捉え、さらに忠が孝よりも上位の概念であるということも、『孟子』のなかの、人を殺した父瞽瞍のために天下を棄てて孝を選択する舜を肯定する場面108を思い出せば、本来の儒教を離れた日本的な解釈というべきものになろう。武内義雄が水戸学を取り上げて、「日本は国家があたかも一大家族のような関係にあって、忠と孝とが一致する。この忠孝一致を力説したのが日本道徳の特徴であり、水戸学の精神である」109といったのは、日本儒教における忠孝の位相を示すものであるといえよう。\n\nところが、その経筵進講から 4 年後の明治 15 年（1882）作成された同じテーマ（弟子入則孝章）の講義草案の内容は、それとは別のニュアンスを伝えている。まさに水戸学を指して、忠のみを優先するその主義を批判したものである。\n\n近世水戸烈公ノ一藩ヲ振起セシ時ニ専ラ尊皇攘夷ノ説ヲ主張シ、忠義節操ヲ以テ人ヲ励マシ、天下其風采ヲ仰キタル時ニ、吾先輩ナル横井小楠カ其学弊ヲ論シテ、専ラ忠義ヲ尚ンテ孝弟ニ本ツカス、其弊必ス気ニ馳セテ国ヲ誤ルコトアラント云テ、⋯⋯横井ノ見識モ実徳ニハ不足モアリテ、悉ク道ニ当リタルニテモ無ケレトモ、其孝弟ニ本ツカサルト云テ、必ス其弊アラント云タルハ、真ニ道ヲ見ルノ識眼ト云ハサルヲ得ス。後ニ水戸ノ君子小人党派相敵シテ遂ニ国ヲ害ヒタルニテ之ヲ鑑ミサルヘカラス110。\n\n元田は小楠とは違って水戸学へのシンパシーを残し続けたといわれるが111、ここでは、小楠が水戸学を批判する「専ラ忠義ヲ尚ンテ孝弟ニ本ツカス」の観点を是認して引きながら、その主義が結局水戸藩の内紛を引き起こした原因になったことを、痛く指摘している。 4 年前の経筵進講での忠中心の立場とは打って変わって、孝をベースに置く考え方の表明である。忠を君臣間の道徳にしても、孝に基づいて忠へと、「夫れ孝は親に事うるに始まり、君に事うるに中し」112というような『孝経』的な本来の儒教に接近しようとするこのような姿勢は、明治 21 年（1888）の『中庸』 20 章の新年進講で「五達道」の順番を説明することからも窺える。\n\n父子有親已下ノ四ツノ道モ素ヨリ重キ道ニテ、特ニ父子ノ親ハ其人ノ生ヲ受ケマスルノ重キヨリ申シマスレハ、父子ノ道ヲ先ニ致シマスレトモ、天下ヲ治メマスルノ上ヨリ申シマスレハ、君臣ノ道立テ始メテ天地モ位シ万物モ育シマスルコトニテ⋯⋯113。\n\n『中庸』の「五達道」では、君臣―父子という順番になっていて、『孟子』の「五倫」が父子を先頭に置くこととは違う。この言葉はその理由を述べたもので、君臣の忠を先に出しているのは、「天下ヲ治メマスルノ上ヨリ」という政治的な目的によるものだという。さらに、明治 15 年（1982）に頒布された『幼学綱要』が、「孝行」を「人倫ノ最大義トス」として一番目に置き、「臣ノ忠節」を「子ノ孝行ニ並ベテ、人倫ノ最大義トス」として二番目に配置したのも114、本来の儒教を考慮したものと把捉できよう。\n\n要するに、元田は「皇道の訓解」゠「我朝ノ伝書」として、その中身を日本的な固有性が色濃い忠孝の徳目によって表象していたが、そうしたなかでも、儒教と「道を同ふす」、もう一つ別の言葉を引けば、「孔子の教、彛倫道徳を主として、天下国家を経綸し、我が邦の道と同じきを以て」115という彼の観念の大前提のもとで、儒教と日本の道（皇道）を調和する姿勢を保持していたのである。それはあるいは、「皇道の訓解」といって、奥深くて知り難い皇道を教えやすい儒教によって補完するという意味を越えて、元田の思考ではまず第一に儒教という絶対的・包括的なスタンダードがあり、その限りにおいて皇道の意義づけを行っていた、といっていいかもしれない。\n\n\n四　結び―時代錯誤の儒学者\n\n以上、元田における革命論と血統論の認識、「皇道の訓解」の意味という二つのテーマを中心に、儒教に加えた皇道主義的言説の思想的座標を検討した。元田の思想営為は、普遍的な儒教に比重を置く皇道主義的儒教なのか、それとも日本の特殊性が重視される儒教的皇道主義とみるべきかという両面を掲げながら、結果的に、前者の儒教中心主義に沿って議論を進めてきたという感も否めない。実を言うと、元田の資料のいたるところで目に付く「堯舜」（政治）を称賛し、その政治が目的となる王道政治・愛民政治を志向する言葉に接して、本稿の着想の段階からそういう方向を考えていたといっていい。\n\nこれまですでに堯舜に言及する元田の言説を取り上げてきたが、ここで改めて直接的に堯舜の政治を目指そうとする元田の言葉を探してみれば、\n\n蓋堯舜ノ君臣ヲ以テ密ニ当世ニ望ムナリ116。（明治 4 年）\n\n今日御講書ノ始ニ此堯典ヲ講スルモ、畢竟堯舜ノ道ガ即今日現実ノ御目的ト相成訳ニテ⋯⋯今日人君タルノ道ヲ尽サント思召サルヽ上ハ、堯舜ヲ御目的ニ遊サルヽヨリ外ハ是ナキコトナリ117。（明治 5 年）\n\n陛下ハ張飛ヲ愛ス。張飛ノ声ハ万人ニ超ヘタリ。今元田ハ堯舜ヲ以テ陛下ニ望ミ奉ル118。（明治 5 年）\n\n当世ニテモ法ヲ取ラセラレマスルニハ、此堯舜ヲ御目的ト遊サレマシテ然ルヘキコトト存上マスル119。（明治 12 年）\n\n堯舜天下ヲ治ムルノ道ヲ知テ、之ヲ実際今日己レニ得テ、之ヲ行フ120。（明治 16 年）\n\nなどがある。明治 4 年の宮中出仕に際して三条実美との面接時のやりとりの言葉から、明 16 年の「書経堯典講義」の言葉まで、ここに挙げたものだけでも、元田の堯舜政治にあこがれる一貫した態度を確認することができる。しかも、その態度は儒学者一般の単なる口癖的な復古調の願望ではなく、本気で「当世」「今日」における実現を目指すものであった。\n\n「誠意正心ノ実心術ノ微ヨリ工夫ヲ下シ⋯⋯治国安民ノ道理利用厚生ノ本ヲ敦ク」121するという、小楠とともに熊本藩での講学時代に築き上げた「実学」の理念に基づく、堯舜を含む言葉で代えていえば「堯舜ノ内ヨリ外ニ及フノ教化」122となる道徳政治の具現を、明治前期の「当世」において真剣に取り組んでいたのである。\n\nそして、元田が堯舜を借りて「当世」に実現しようとしていた具体的な政治が、人民を愛し優先する王道政治・愛民政治であった。\n\n唯人君宰相心術ヲ正シ、品行ヲ修メ、⋯⋯万機ノ政ヲ為ス時ハ、法制ヲ設ルモ、禁令ヲ出スモ、刑罰ヲ施スモ、教化ヲ布クモ、皆民ヲ愛スルノ余リニ発シ、石室ヲ造ルモ、鉄道ヲ開クモ、一利ヲ興スモ、一害ヲ除クモ、総テ民ヲ先ニシテ己ヲ後ニシ、所謂人ニ忍ヒサルノ心ヲ以テ人ニ忍ヒサルノ政ヲ行フ123。\n\n蓋シ人君ノ大徳ハ仁ニ止マリテ、治道ハ人ヲ愛スルニ在ルノミ。若シ人ヲ愛スルノ心至ラサル時ハ、事業大ナリト雖、国家富強ナリト雖トモ、天職ノ仁ニ悖レハ其余ハ観ルニ足ラス。今陸海軍ヲ皇張スルモ人ヲ愛スルカ為メナリ。憲法民法ヲ布クモ人ヲ愛スルニ因テナリ。教育勧業モ人ヲ愛スルカ為メナリ。鉄道電信モ人ヲ愛スルニ由テナリ。凡ソ国家ノ施ス所、一トシテ人ヲ愛スル事ニ非サルコトナシ。若シ人ヲ愛スルノ心ヨリ出テスシテ徒ニ事業ヲ盛ニシテ、欧州ノ文明ト競争セント欲スルカ如クナラハ、則是国家ニ長タルノ人ノ心ニ非スシテ、民心亦服スヘカラス124。\n\nこれが、儒学者たる侍講として、元田の目指す政治の最終目的地ではなかったろうか。しかし、新国家の青写真がまだ何も決まっていなかった小楠の時代とは違って、西洋近代化が積極的に進められていた時代のなかで、「道徳ハ政事法律ノ本体、政事法律ハ道徳中ノ手足」125というような堯舜の道徳政治が地歩を広げる余地はなかった。明治 12 年（1879）に元田が中心となって進めていた「君徳培養ヲ主トスル」126という「侍補制」が廃止に追い込まれたことは、その辺の事情を象徴的に伝えている。その意味で元田の堯舜宣揚と儒教中心主義は保守反動であり、それが目指す王道政治の理想的な意義はともかく、「時代錯誤」的なものでしかなかった。\n\n元田の儒教思想における時代錯誤的な要素は、探そうとすればいくらでもあろう。君主（天皇）と人民の関係を父母と子の間に比肩して、人民の君主への従属的関係として設定したことも、その一つである。\n\n舜ノ天下人民ヲ治メマスルハ、丁度父母ノ子ヲ愛シ育テマスルヨウニコサリマス故ニ、子ノ望ミマスル所、子ノ言ハント欲シマスル所ニ、舜親カラ先立マシテ、四方ノ門ヲ開キ、四方ノ耳目ヲ明ケマシテ、サア言ヘ、サア願ヘ、カヤウニ致シテ遣ハサフ、カウ致スカ宜シイト引起シ引起シ致シマスルコトテ、中々後世西洋ナドノヤウニ下カチニ人民ノ驕リマスルヤウノコトハ、聊之ナキコトテコサリマスル故ニ、先ニモ神武天皇ノ御心ト同シ心ト申上マスル如ク⋯⋯127。\n\n先に引用した明治 12 年講義の前文で、当世において堯舜を倣う理由について述べたものである。西洋の「下カチニ人民ノ驕リ」のこととは対照的に、日本では最初から舜の政治のように、天皇と人民は親子関係としてあった（あるべき）という。また元田においてその関係は、「人君ヨリ人民ニハ始メヨリ愛育スルト申シマスル天職ノ約信カ備リマシテ、人民ヨリ人君ニハ亦始メヨリ保護ヲ受ケルト申シマスル天分ノ願済ヲ得マシテ居マスル」128とも説かれていて、人君には人民を育てる、人民には人君の保護を受けるという役割ないしあり方が「天職」「天分」として与えられているという。\n\nこうした主張にあっては人民の政治参与、つまり当時の自由民権運動が否定されるのは当然といえば当然のこととなろう。\n\n堯舜ノ道ヲ以テ天下ヲ治メント志スハ人君ノ事ナリ。人臣トシテ其職ニ在ラスシテ、茲ニ志スハ寧ロ位ヲ出ツルノ思ヒニシテ、或ハ民権論者ノ人民参政ノ権アルト云ニマガフト云者モアランカ、是大ナル違ヒナリ。凡ソ此ノ如キ論、皆道理ヲ知ラサル故ニ、当世西洋ノ説ヲ誤認シテ大ナル弊害ヲ生セシナリ129。\n\n天下統治は人君の固有の仕事なので、人臣がそれを僭し、人民が政治に参与することは、西洋の悪影響による「大ナル違ヒ」であると非難している。そして続く言葉では、人々に「仁義五常の性」が具われている側面においては「上下貴賤隔テナキ所」だといいながら、「然トモ其事ニ於テハ君臣ノ分アリ、大臣小臣ノ別アリ、士農工商ノ等アリ、出処進退ノ異ナルアリ。故ニ臣民トシテ其位ニアレハ其政ニアツカリ、其位ニアラサレハ其政ニアヅカラス」130と、『論語』の「その位に在らざれば、その政を謀らず」（泰伯）という正名論的な論法に依拠して、自説を正当化している。\n\nさらに、元田がこの延長線上で「君権」を強調することも、時代錯誤的な儒学者の一側面となる。\n\n憲法を建てゝ、其の自由を与へ給ひ、各種の法律を設けて不法を制し給ふも、総て君徳中の事にて、民の権利は皆君権にありて、君権は、君徳の勢力範囲を云ふなり。⋯⋯万の法律、一つも君主の大権に帰せざるはなし。我臣民たる者、賜ふ所の権利を拝取して、誰か敢えて君上に向ひ、民権を唱ふ者あるべんや131。\n\n憲法を含めてすべての法律は最終的に「君権」に帰属するものなので、人民はただ君権によって与えられた権利を「拝取」さえすればいいという。人民が自由に政治に参加する、今日的な「民主」という観念は徹底的に無視されているのである132。\n\n以上、作為的な制度論より自然的な心性・道徳論の重視、君主と人民の関係を家族関係になぞらえて服従的な性格を持たせたこと、君権の強調と民権の軽視といった、元田の儒教思想におけるいくつかの時代錯誤的な主張を取り上げたが、ここで注意したいのは、それは彼が儒教思想を誤解したり間違って理解したことによるものではなく、むしろ誰よりもまじめな儒学者・朱子学者であったことに起因する、ということである。儒教の政治思想には、元田がよく進講の主題とする『書経』が典拠の「民は惟れ邦の本なり」（夏書）という「民本」はあるが、上述の「民主」概念は本来欠落されているからである。\n\nそして、元田の時代錯誤的な主張は、彼が皇道主義者であったことによるものでもないことも指摘しておきたい。元田以後、いわゆる「家族制国家」のもとで、人民（臣民）の天皇への忠誠の責務がやたらに肥大していった状況を想起すれば133、上述の時代錯誤の引用で傍点をつけたところ―「愛育」「親カラ先立」「君徳」―だけをみても、元田は儒教の教えに従って治者側の道徳的な涵養・率先を絶えず要求していたからである。その点で元田の儒教思想は、後日の「本邦固有の皇道及国体に醇化したる儒教」134を標榜する露骨な儒教的皇道主義とは違うものがあり、本稿は主としてそのことを検討したものである。\n\n\nデータ利用可能性\n\n本稿の研究結果の基礎となるデータは、すべて本論文中に示されており、追加のソースデータは必要とされていない。", "appendix": "Footnotes\n\n1 元田竹彦・海後宗臣編『元田永孚文書』第二巻（元田文書研究会、1969 年）、『経筵論語進講録』、109 頁。以下、同書は巻数表示を漢数字によって示す。なお、引用文の旧字体は常用漢字に変え、適宜句読点を加えた。\n\n2 元田竹彦・海後宗臣編『元田永孚文書』第三巻（元田文書研究会、1970 年）、『論議講義』、231 頁。以下、同書は巻数表示を漢数字によって示す。この他に「神教の注解」（海後宗臣『元田永孚』日本教育先哲叢書 19 、文教書院、1942 年、190 頁）という表現もある。\n\n3 沼田哲『元田永孚と明治国家―明治保守主義と儒教的理想主義』（吉川弘文館、2005）。序論の冒頭で「元田永孚という熊本藩出身の儒学者が居る」（1 頁）といい、注においてこの問題を提起している（17 頁）。\n\n4 沼田は、小楠と違う元田の思想場面を、水戸学（国体論的考え方）への親近感を持続していたこと（同書、53～54 頁）、小楠が「堯舜三代之道」を政道のモデルとして掲げていたに対して、元田は「我国上古の天祖神聖の道」への回帰を主張したことなどを挙げている（162 頁）。純粋な儒学者という点からすれば、小楠より元田が遠く離れているということはいえよう。\n\n5 久木幸男「明治儒教と教育―1880 年代を中心に―」（『横浜国立大学教育紀要』 28 、1988 年） 251～259 頁。その失敗の主因は「元田が儒教とは全く異質の「天皇尊崇」を無理に組み入れ、「天皇尊崇」を中心に儒教道徳を再編しようとした」ことにあるという（259 頁）。\n\n6 同論文、25 頁。\n\n7 岩井忠熊『明治国家主義思想史研究』（青木書店、1972 年） 59～66 頁。\n\n8 松浦玲「日本における儒教型理想主義の終焉（一）」（『思想』 571 号、岩波書店、1972 年 1 月）、「実学と儒教思想―日本における儒教型理想主義の終焉（四）」（『思想』 630 号、岩波書店、1976 年 12 月）。\n\n9 松浦、前掲「日本における儒教型理想主義の終焉（一）」、38 頁。\n\n10 松浦、前掲「実学と儒教思想」、106 頁。\n\n11 土屋忠雄『明治前期教育政策史の研究』（講談社、1962 年） 360 頁。\n\n12 海後、前掲『元田永孚』、73 頁。\n\n13 同書、86 頁。\n\n14 松浦、前掲「実学と儒教国家」、107 頁。\n\n15 同論文、109 頁。「要は、君主を、為政者は聖人であるべきだという朱子学的原則の例外としてはならないところにある。君主こそが先頭に立たねばならない」という論法である。\n\n16 元田竹彦・海後宗臣編『元田永孚文書』第一巻（元田文書研究会、1969 年）、『還暦之記』、129 頁。以下、同書は巻数表示を漢数字によって示す。\n\n17 同書、158 頁。\n\n18 同書、126 頁。\n\n19 石田一良は、元田の「教育勅語」を「家族制国家の理念」として把捉し、明治 30・40 年代に至って『明治民法』と穂積八束らによって「本来元田が希望したとおり」になり、この人間型は第二次世界大戦の終了まで日本人の中核を構成したという（『日本思想史講座 6 近代の思想 1 』、雄山閣、1976 年、232 頁）。\n\n20 小倉紀蔵は、元田の使命は道徳を具えた天皇が中心となる儒教的システムを作ることであり、「元田のこのような意図がいかに挫折・変質し、その挫折と変質がいかにその後の日本を規定したか」を分析することが、元田研究の意義であるという（『朱子学化する日本近代』、藤原書店、2012 年、258 頁）。\n\n21 『日本之儒教』（日本儒教宣揚会、1934 年） 204～217 頁。\n\n22 飯島忠夫『日本の儒教』（国体の本義解説叢書、教学局、1937 年） 99～100 頁。\n\n23 同書、102 頁。\n\n24 山鹿素行『中朝事実』、『山鹿素行全集思想篇』第十三巻（岩波書店、1935 年） 42 頁。\n\n25 同前。\n\n26 同書、7 頁。\n\n27 同書、41 頁。\n\n28 松岡仲良『神道学則日本魂』、『近世神道論・前期国学』（日本思想大系、岩波書店、1972 年） 253 頁。\n\n29 同書、260 頁。\n\n30 熊沢蕃山『集義和書』、『熊沢蕃山』（日本思想大系、岩波書店、1971 年） 215 頁。\n\n31 同書、149 頁。\n\n32 同書、153 頁。『孟子』引用の「代をかさねて～天の廃する所なり」についてはまた後述する。\n\n33 蕃山は「帝王の天下」から武家政権に移った原因を、「謙徳を失ひ給ひし故に、天下の権威を失ひ給へりなり。⋯⋯其後王徳をとろへ、国々に我まゝなる者出来て⋯⋯」という（同書、149 頁）。\n\n34 同書、11 頁。後醍醐天皇の余計な政治参与によって「一向武家の世とはなれり」というものである。\n\n35 同書、151 頁。\n\n36 この問題をめぐる蕃山と元田の違いについては、森川輝紀「元田永孚と教学論」（『埼玉大学紀要教育学部』 59 （1）、2010 年、140～141 頁）から示唆を得た。\n\n37 元田・海後、前掲『元田永孚文書』一、『還暦之記』、129 頁。これは明治 4 年、初出仕の際の言葉。\n\n38 沼田哲・元田竹彦編『元田永孚関係文書』（近代日本史資料選書 14 、山川出版社、1985 年） 148 頁。明治 11 年、下津休也に与えた手紙の一節。\n\n39 元田・海後、前掲『元田永孚文書』一、『当官日箚』、327 頁。明治 11 年の言葉。\n\n40 同書、『還暦之記』、128 頁。\n\n41 海後、前掲『元田永孚』、「君徳輔導の上言」、175 頁。\n\n42 同書、「教育大意私議」、193 頁。\n\n43 同書、「国憲大綱」、145 頁。\n\n44 同書、「教育大旨」、147 頁。\n\n45 同書、「君徳輔導の上言」、174 頁。\n\n46 徳富猪一郎『増補元田先生進講録』、「元田東野翁」（明治書院、1910 年） 3 頁。山室信一はこの「臨機応変者」の言葉を挙げながら、元田の立場を「小楠の嗚乎血統論是豈天理順との堡塁を守るにはあまりに軽俊にすぎたし、自己のよって立つ地地盤の何たるかが見えすぎていた」という（「天皇の聖別化と国「教」論」、『近代熊本』 21 号、熊本近代史研究会、1981 年、3 頁）。\n\n47 元田・海後、前掲『元田永孚文書』二、『新年講書始進講録』（以下、新年進講録に略す） 62 頁。\n\n48 黃宗羲『明夷待訪錄』原君（台湾中華書局印行、1969 年） 3 頁。\n\n49 同書、2 頁。\n\n50 『孟子』万章下（徐徳明校点『四書章句集注』、『朱子全書』第陸冊、上海古籍出版社・安徽教育出版社、2002 年） 376 頁。\n\n51 司馬光『伝家集』巻 73 「疑孟」（『影印文淵閣四庫全書』 1094 冊、台湾商務印書館、1986 年） 666 頁。\n\n52 『孟子』万章上、前掲『四書章句集注』、376 頁。\n\n53 『孟子』滕文公下、前掲『四書章句集注』、331 頁。\n\n54 『孟子集注』梁恵王下、前掲『四書章句集注』、269 頁。\n\n55 安炳周『儒教의 民本思想』（成均館大学校出版部、1987 年） 54 頁。\n\n56 ちなみに、「「沼山閑居雑詩」のなかの「嗚呼血統論是豈天理順」という一句によって、血統論に反対したといわれている小楠も、元田との談話の中では武王の放伐を認めていなかった。「文王の時既に三分の二を保ちたれば武王に到りて殷益小弱なる事知る可なり。此時に当りて時勢を云ふにも何ぞ殷を伐つに及ばんや。文王の如く只徳政を修め民を恤み、紂王は紂王にして存し置かば自ら衰へて終る可し。然るを我より事を起し人臣の道を失ふて数世連綿の国君を我手にかけて亡す事さてもさても道を得ざる事と伯夷は真誠明了に存じ込みしより馬を叩て諫たり」（山崎正董『横井小楠遺稿』、『沼山閑話』、日新書院、1942 年、925 頁）。一方で、元田は小楠の「嗚呼血統論」の詩に対して、「千百年来の活眼、天理の実、易道の本に達する者にあらざれば言ふ能はず」という、大いに肯定するような評価を与えていた（山崎正董『横井小楠伝記篇』、明治書院、1938 年、354 頁）。要するに、この問題をめぐる彼らの見解も一貫しているとはいえない状況で、一方的な裁断は無意味であるということを伝えているのであろう。\n\n57 元田・海後、前掲『元田永孚文書』一、『還暦之記』、100 頁。小楠の「又曰吾兄利口ヲ止メヨ、利口誤ル所アラン。而シテ先生自ラ云、我（゠元田）ハ利口者ナリ、人ノ及ハサル所」ということに対して、元田は「余自顧ルニ利口者タルコトヲ覚ヘス。⋯⋯先生ノ能ク余カ才質ヲ知ル所ナリト感服スルナリ」と受け止めていた。\n\n58 たとえば、小楠は『国是三論』のなかでワシントンの業績を戦争終息、大統領の退任、共和政治などとして挙げながら、「殆三代の治教に符合するに至る」と評価していた（山崎、前掲『横井小楠遺稿』、40 頁）。\n\n59 この二文が著述された明確な時期は不明であるが、沼田哲の整理によれば、慶応元年（1865）以前の文として並べられている（「「元田永孚関係文書補遺」並びに「元田永孚文書目録」」、『青山史学』 10 号、1988 年、133 頁）。なお、その全文は、原漢文を書き下し文にして前掲の山崎『横井小楠伝記』に収録されている。\n\n60 山崎、前掲『横井小楠伝記篇』、1142～1143 頁。\n\n61 同書、1130～1131 頁。\n\n62 同書、1131 頁。\n\n63 元田・海後、前掲『元田永孚文書』三、『書経講義』、74 頁。\n\n64 元田・海後、前掲『元田永孚文書』一、『還暦之記』、68 頁。元田が小楠の開国論とワシントン論を聞いて述べたもので、「安政乙卯ノ年」（1855）の記録があり、元田 30 代前半の時期の言葉である。\n\n65 出仕後の言葉としては、明治 11 年（1878）の「論語為政首章」の進講草案に「華盛頓の如きは、全く徳を以て国を興し、業を創めしなり」（海後、前掲『元田永孚』、137 頁）というのがある。ただし、『元田永孚文書』二に収録されている『経筵論語進講録』の該当講義案には、この部分が省略されて見当たらない。\n\n66 元田・海後、前掲『元田永孚文書』二、『新年進講録』、85 頁。\n\n67 同書、86 頁。\n\n68 同書、71 頁。これは明治 18 年（1885）、元田 68 歳の『書経』虞書益稷の末節の新年進講の一節。\n\n69 天皇の現実政治への参与を否定し、君徳培養の帝王学の必要性も認めない蕃山の天皇論は、皮肉にも明治期において元田的な儒教主義に反対した福沢諭吉の言説に重ねられる。福沢は「帝室は万機を統るものなり、万機に当るものに非ず」（『帝室論』、再版『福沢諭吉全集』第 5 巻、岩波書店、1970 年、263 頁）として、「帝室の神聖は政治社外の高処に止まりて広く人情の世界に臨み、其余徳を道理部内に及ぼして全国の空気を緩和せんこと」（『尊王論』、前掲再版『福沢諭吉全集』第 6 巻、18 頁）を主張した。\n\n70 海後、前掲『元田永孚』、「教育大義私議」、189～190 頁。\n\n71 「智（知）仁勇」の四書での出典は、「子曰、知者不惑、仁者不憂、勇者不懼」（『論語』子罕、前掲『四書章句集注』、147 頁）、「知仁勇三者天下之達徳也」（『中庸章句』 20 章、前掲『四書章句集注』、45 頁）。\n\n72 「唯天下至聖、為能聰明睿知、足以有臨也、寬裕温柔、足以有容也、發強剛毅、足以有執也」。\n\n73 元田・海後、前掲『元田永孚文書』二、『新年進講録』、64 頁。\n\n74 北畠親房『神皇正統記・増鏡』（日本古典文学大系 87 、岩波書店、1965 年） 60～61 頁。\n\n75 親房の智仁勇への言及は『東家秘伝』にみえ、「解曰、如レ玉ニシテ曲妙ナルハ、柔順ノ心ヲ表シ給也、仁也。如鏡にして分明ナルハ、正直ノ心ヲ表シ給也、心ノ本元也。如剣剛利ナルハ、決断心ヲ表シ給也、智也、勇也。尚書ニハ、剛・柔・正直ノ三徳トモ云、礼記ニハ、智仁勇ノ逹徳トモ云、其義皆一也」（『神道大系論説編十八北畠親房（上）』、財団法人神道大系編纂会、1991 年、355 頁）とある。しかしここでも玉゠仁、鏡゠正直、剣゠智・勇として、元田のような配当とはずれが認められる。\n\n76 和辻哲郎『日本倫理思想史』下（岩波書店、1952 年） 52 頁。\n\n77 熊沢蕃山『大学或問』、前掲『熊沢蕃山』、448 頁。\n\n78 熊沢蕃山『神道大義』、『蕃山全集』第五冊（蕃山全集刊行会、1942 年） 13 頁。\n\n79 同書、14 頁。\n\n80 同書、239 頁。\n\n81 和辻、前掲『日本倫理思想史』下、447 頁。\n\n82 山崎闇斎『大和小学』、『山崎闇斎』上（日本教育思想大系、日本図書センター、1979 年） 18 頁。\n\n83 「粹然たる」という形容は井上哲次郎の元田評価を借りたものである。井上は元田の儒教思想理解について、「決して仁斎、益軒、鳩巣等の下にあらず、⋯⋯が如きは粹然たる儒教の代表者といふを得べきなり」と評価していた（『日本朱子学派之哲学』、冨山房、1905 年、566 頁）。江戸時代に鏡・玉・剣を儒教の智仁勇に対応させた儒者としては、林羅山「此三ノ内証ハ鏡ハ智也、玉ハ仁、剣ハ勇、智仁勇ノ徳ヲ一心ニタモツ義也」（『神道伝授』、『近世神道論・前記国学』、日本思想大系、岩波書店、1972 年、12 頁）、山鹿素行「玉は以て温仁の徳を表はすべく、鏡は以て致格の知を表はすべく、剣は以て決断の勇を表はすべし」（『中朝事実』、前掲『山鹿素行全集思想篇』第十三巻、45 頁）などが挙げられる。\n\n84 『中庸』、前掲『四書章句集注』、59 頁、校勘記〔一〕。\n\n85 元田・海後、前掲『元田永孚文書』二、『新年進講録』、69 頁。\n\n86 海後、前掲『元田永孚』、「教育大義私議」、193～194 頁。\n\n87 『孟子集注』告子上、前掲『四書章句集注』、400 頁。\n\n88 とはいえ、神器の徳目の所有を一般化する解釈は先輩の思想家にもあり、元田が引用する蕃山もそれに言及していた。「三種の象は孔門伝授の心法なり」（『大学或問』下冊、前掲『熊沢蕃山』、449 頁）として、『中庸章句』の朱子注の言葉（孔門伝授の心法）を引いて、「三種の神器は禁中におはしますといへども、其の象の心法は、上天子より下庶人に至まで、身を修るを以て本とするの義なり」（『三輪物語』、前掲『蕃山全集』第五冊、13 頁）というものである。しかし、その理解は原論的な言及に止まり、元田の徹底した理解には及ばない。前述した蕃山と元田の儒教へのコミットの差異がここにも出ているといえよう。\n\n89 元田の「国教」という概念は、明治 12 年（1879）に元田が起草し発表された「教育大旨」に対して、伊藤が「教育議」を奏上し、また元田が反論の形式で「教育附議」を著したことから顕在化した言葉である。元田が「祖宗ノ訓典ニ基ツキ」「祖訓ヲ敬承」することを「国教」とし、その中身を儒教に充当させることを提案したことに対して、伊藤はその儒教中心の教育方針を拒否したのであった（『教育勅語渙発関係資料集』第一巻、国民精神文化研究所、1938 年）。\n\n90 海後、前掲『元田永孚』、「国教論」、204 頁。\n\n91 元田・海後、前掲『元田永孚文書』二、『新年進講録』、70 頁。\n\n92 海後、前掲『元田永孚』、「国教論」、206 頁。\n\n93 『教育勅語渙発関係資料集』第二巻（国民精神文化研究所、1939 年） 297 頁。原漢文。海後宗臣よれば、明治 17 年に認められた「国教論」は 3 種あるというが（海後、前掲『元田永孚』、115 頁）、この漢文体の文章には七月の記が付いている。伊藤に提出したものには 8 月の記がみえる。\n\n94 元田・海後、前掲『元田永孚文書』三、『論語講義』、341 頁。\n\n95 さらには、「教育勅語」の末尾の「朕爾臣民ト俱ニ拳々服膺シテ」という句も、そこに示されている諸徳目を天皇も実践する義務があることを示している（苅部直『日本思想史の名著 30 』、ちくま新書、2018 年、198 頁）。\n\n96 元田・海後、前掲『元田永孚文書』二、『経筵論語進講録』、121 頁。\n\n97 海後、前掲『元田永孚』、「為学之要」、173 頁。\n\n98 元田・海後、前掲『元田永孚文書』三、『論語講義』、231 頁。\n\n99 徳富、前掲『増補元田先生進講録』、「論語学而章」、1 頁。\n\n100 北畠親房、前掲『神皇正統記』、79 頁。\n\n101 山崎闇斎、前掲『大和小学』、4 頁。\n\n102 山鹿素行、前掲『中朝事実』、62 頁。\n\n103 伊藤仁斎『童子問』、『近世思想家文集』（日本古典文学大系、岩波書店、1966 年） 58 頁。\n\n104 伊藤仁斎『論語古義』総論、『「論語」名家註釈書（一）』（「論語」叢書、大空社、2011 年） 3 頁。\n\n105 元田・海後、前掲『元田永孚文書』二、『経筵論語進講録』、157 頁。\n\n106 同書、158 頁。\n\n107 『論語』学而、前掲『四書章句集注』、69 頁。\n\n108 『孟子』尽心上、前掲『四書章句集注』、437 頁。\n\n109 武内義雄「日本の儒教」、『武内義雄全集』 4 巻（角川書店、1978 年） 114 頁。\n\n110 元田・海後、前掲『元田永孚文書』三、『論語講義』、280 頁。\n\n111 代表的に、沼田、前掲『元田永孚と明治国家』、53～54 頁。\n\n112 『孝経』開宗明義章（『諸子集成補編一』、四川人民出版社、1997 年） 4 頁。\n\n113 元田・海後、前掲『元田永孚文書』二、『新年進講録』、90 頁。\n\n114 前掲『教育勅語渙発関係資料集』第一巻、33 ・ 44 頁。\n\n115 元田・海後、前掲『元田永孚文書』二、『経筵論語進講録』、158 頁。\n\n116 元田・海後、前掲『元田永孚文書』一、『還暦之記』、127 頁。\n\n117 元田・海後、前掲『元田永孚文書』二、『新年進講録』、3 ・ 5 頁。\n\n118 元田・海後、前掲『元田永孚文書』一、『還暦之記』、135 頁。\n\n119 元田・海後、前掲『元田永孚文書』二、『新年進講録』、39 頁。\n\n120 元田・海後、前掲『元田永孚文書』三、『書経講義』、4 頁。\n\n121 元田・海後、前掲『元田永孚文書』一、『還暦之記』、27 頁。\n\n122 元田・海後、前掲『元田永孚文書』二、『新年進講録』、43 頁。\n\n123 同書、13 頁。\n\n124 元田・海後、前掲『元田永孚文書』二、『経筵論語進講録』、143 頁。\n\n125 元田・海後、前掲『元田永孚文書』二、『新年進講録』、92 頁。\n\n126 元田・海後、前掲『元田永孚文書』一、『古稀之記』、179 頁。\n\n127 元田・海後、前掲『元田永孚文書』二、『新年進講録』、38～39 頁。\n\n128 同書、29 頁。\n\n129 元田・海後、前掲『元田永孚文書』三、『書経講義』、4 頁。\n\n130 同上。\n\n131 海後、前掲『元田永孚』、「論語為政首章」、139 頁。\n\n132 苅部直は、元田の「自由」「権利」の概念を検討し、彼の「道徳社会」では「個々人の「不羈自由」は完全に封殺される」と指摘している（『歴史という皮膚』、岩波書店、2011 年、224 頁）。\n\n133 たとえば『国体の本義』（文部省、1937 年）に、「抑々我が国は皇室を宗家とし奉り、天皇を古今に亙る中心と仰ぐ君民一体の一大家族国家である」（38）として、それゆえに「即ち我等は、生まれながらにして天皇に奉仕し、皇国の道を行ずるものであって、我等臣民のかかる本質を有することは、全く自然に出づるのである」（32）とある。\n\n134 前掲『日本之儒教』、例言。" }	[ { "id": "83015", "date": "13 Apr 2021", "name": "Ryoko Shimokawa", "expertise": [ "Reviewer Expertise 日本思想史" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n本論文は、明治天皇の侍講となり、『教育勅語』の起草に関わるなど、明治の言論界で活躍した元田永孚の皇道主義的言説を分析し、その儒教思想の構造を明らかにしたものである。彼は、革命論を是認せず、血統論を既定の事実として王道政治実現の主体である君主の徳の涵養をめざした儒者の標準的思考の持ち主だった。さらに皇道（神道）の意味を、朱子学の文献、例えば『中庸章句』の三達徳の概念を用い論理づけようとした。北畠親房や熊沢蕃山がもっていた曖昧さを排除し、より儒教的解釈に徹したのである。そして、三種神器の徳目を天皇に限定せず、人々の普遍的性とする朱子学的本性論を展開した。各自の努力でその徳を極大化して修身治国平天下をめざすという主張は、朱子の『大学』解釈を踏まえたものである。\n元田の思想は、国教として天皇を神聖視し皇道を絶対化したものだと言われるが、実際には儒教の政治論の伝統的教義にのっとっているのである。忠孝の徳について、日本では孝よりも忠を重視し、それが天皇中心思想につながると言われてきたが、元田永孚は、忠を強調した水戸学を批判し、孝経的な本来の儒教に近づけようとした。本論文は、結論として、彼がめざしたものを、堯舜の治政の再来、人民を愛し優先する儒教的王道政治と指摘する。幕末から明治の皇道的な思想の系譜の中で語られる元田の論理を、東アジア全体の儒教思想史の中に位置づけた本論文の結論は十分に説得力がある。\nただ、元田の儒教思想を評価する際に、朱子学的論理と西洋的自然法思想との本質的類似性に着目し、天賦人権論の文脈に儒教を援用した中江兆民との違いにも言及があれば、明治期における儒教思想の果たした役割がより明確になったと思われる。けれども、元田の儒教思想の構造を明らかにした本論文は日本思想史研究、東アジア儒教史研究に大きく寄与するものと考えられる。\n\n本研究は明確かつ正確に提示されたものであり、最新の文献を引用していますか。はい\n\n研究設計は適切で学術的価値がありますか。はい\n\n方法と分析について第三者による再現が可能となるよう十分な詳細が提示されていますか。はい\n\n（該当する場合は要回答）統計分析および解釈は適切ですか。対象外（統計を使っていない\n\n結果の基礎となるソースデータはすべて入手可能で再現性を十全に保証していますか。はい\n\n結論は結果により妥当な裏付けを得ていますか。はい", "responses": [ { "c_id": "6608", "date": "27 Apr 2021", "name": "Seogin Eom", "role": "Author Response", "response": "新学期の慌ただしい時期に、詳しく読んで下さり、また評価していただき、真にありがとうございました。ご指摘いただいた中江兆民との比較は、二人の民権をめぐる議論も含めて、別のテーマとしても興味深いものがあるかと思います。機会があれば挑戦してみたいと思います。" } ] }, { "id": "83014", "date": "22 Apr 2021", "name": "Ryu Kataoka", "expertise": [ "Reviewer Expertise 片岡龍（東アジア思想史）増田友哉（近世日本国学、皇国主義思想）" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n○総評　本論文は従来、皇道主義と儒教思想との間で揺れのあった、元田永孚の思想的評価を、元田の皇道主義的言説を分析することによって、「普遍的な儒教に比重を置く皇道主義的儒教」として確定したものである。その分析は優に学術的水準を満たしており、結論も妥当である。　強いて言えば、皇道主義と儒教思想かという二項対立的な問題設定の枠組み自体を突破しうるだけの鋭い分析がなされながらも、結論が結局は枠組みの中での評価の精緻化にとどまっている点が、惜しまれる。思想史的評価に際しては、言説分析にとどまらず、元田の社会的実践との関係も考慮に入れる必要があるのではないか。　以下、細にわたるが、確認を希望する点を列挙しておく。\n○誤字脱字の確認を要する箇所　・注21の引用文\n\n「異帯なる」→「偉大なる」？　・注47の引用文\n\n「世ノ三」→「世ノミ」？　・注48の引用文他複数個所\n\n「傑」→「桀」？　・注131の引用文\n\n「あるべんや」→「あるべけんや」　・九頁四段落\n\n「加担していたなと」→「加担していたなど」？\n○日本語（訓読含む）表現としての妥当性の検討を要する箇所　・三頁四段落\n\n「継ぎ手」→「継承者」？　・注18直後\n\n「懐述」→「述懐」？　・注48の引用文\n\n「源を小儒に導くに非らずや」→「源を小儒に導かるるに非ずや」？\n\n○文意が曖昧なため、表現の修正の検討を要する箇所　・三頁二段落　「すこし大げさに聞こえるかもしれない理由によるものである。」　→(1)何の理由か、(2)誰に「大げさに聞こえる」のかが、一読して分かるような表現の工夫　※総じて三頁二段落の全体が、導入として迂遠に感じる。\n○内容に関する確認を要する箇所　・「純粋でない儒教」（五頁一段落）、「まじめな儒学者」（一七頁最終段落）という用語はなにをもって「純粋」「まじめ」とするのか、論者の価値観が混入していることにならないか？　・五頁三段落　「革命論は中国と韓国の歴史における王朝交替の正当性を担保」\n\n→韓国の場合、そう言い切れるか？　・十四頁～十五頁にかけての段落\n\n→忠孝一致を単純に日本的と断定できるか？　たとえば『礼記』祭統に「忠臣以事其君、孝子以事其親、其本一也」。　もちろん、『論語集注』（八佾「君使臣以礼、臣事君以忠」）に「尹氏曰、君臣以義合者也。故君使臣以礼、則臣事君以忠」、『朱子語類』巻十三に「父子兄弟夫婦、皆是天理自然、人皆莫不自知愛敬。君臣雖亦是天理、然是義合。世之人便自易得苟且。故須於此説『忠』、卻是就不足処説」とあるように、「天理」である孝と「義合」である忠との区別を意識した上で、だろうが。\n\n本研究は明確かつ正確に提示されたものであり、最新の文献を引用していますか。はい\n\n研究設計は適切で学術的価値がありますか。はい\n\n方法と分析について第三者による再現が可能となるよう十分な詳細が提示されていますか。はい\n\n（該当する場合は要回答）統計分析および解釈は適切ですか。対象外（統計を使っていない\n\n結果の基礎となるソースデータはすべて入手可能で再現性を十全に保証していますか。はい\n\n結論は結果により妥当な裏付けを得ていますか。はい", "responses": [ { "c_id": "6609", "date": "07 May 2021", "name": "Seogin Eom", "role": "Author Response", "response": "新学期のご多忙の時期に、ご丁寧に読んで下さり、適切なご指摘・ご教示いただきまして、心より感謝申し上げます。元田の思想史的評価においては彼の社会的実践の側面も考慮に入れるべきだというご指摘には、筆者も痛く共感しているところです。今回はそこまで論ずる余裕はなかったのですが、別の機会に譲りたいと思います。改訂版にてご指摘のあった曖昧な文章や脱字誤字等を訂正しました。内容確認の部分については、簡単ながら補足説明いたします。・「純粋でない儒教」については、「純粋」か「不純」かのような価値的な区別ではなく、儒学者として儒教的言説のほかに、それとは別の性質の言説もまじえて表現したという意味で用いたものです。あえていうならば、壇君神話のようなものを儒教言説と一緒に提出する、といった習合的な傾向はほとんどみられない韓国儒教との比較的観点はあったかと思います。・「まじめな儒学者」については、強調的表現です。・「革命論は中国と韓国の歴史における王朝交替の正当性を担保。→韓国の場合、そう言い切れるか？」については、新羅最後の敬順王が高麗の王建に自ら帰順し、高麗と朝鮮の王朝交替のときに、「禅譲」の形式を取っていたことを、念頭に置いたものです。・「忠孝一致を単純に日本的と断定できるか？」についてはご教示のような言説（『論語集注』『朱子語類』）にもよりますが、元田自身がそれを「世界無比」として持ち出していたことを踏まえました。" } ] } ]	1	https://f1000research.com/articles/10-272
https://f1000research.com/articles/10-352/v1	06 May 21	{ "type": "Case Report", "title": "Case Report: A five-year follow up after pediatric renal transplantation using flow cytometry crossmatch and HLA immunophenotyping based on DNA for screening test", "authors": [ "Johanes Dwi Meiyanto", "Besut Daryanto", "Kurnia Penta Seputra", "Kurnia Penta Seputra" ], "abstract": "Background: There are three methods for renal replacement therapy for end stage chronic kidney disease; dialysis, continuous ambulatory peritoneal dialysis, and renal transplantation which is the best because of the least morbidity rate, the best survival rates, the best quality of life, and the best improvement in activities of daily living. In the field, flow cytometry serves a well-established role in pre- and post-transplant crossmatching, and if it is combined with human leukocyte antigen (HLA) immunophenotyping based on DNA, it will produce a more sensitive prediction of the chronic graft rejection compared to complement-dependent cytotoxicity crossmatching and can eliminate irrelevant antibody (IgM). This is the first experience using this method in our hospital. The survival rate at one, five and ten years has been shown to be 99%, 97% and 96%, respectively; therefore, we wanted to find out the five year follow up of the patient. Case presentation: We evaluated a 20-year-old female with a history of pediatric renal transplantation five years previously due to end stage renal disease caused by bilateral parenchymatous renal disease. She had a history of hypertension since December 2014 and underwent hemodialysis for three months. The transplantation took place in March 2015. A kidney from her mother was transplanted to recipient using end-to-side anastomoses. After five years, the patient was routinely monitored at the urology clinic, with creatinine serum results between 1.5 and 2 mg/dL, urea and electrolyte serum levels within normal limits and she could resume normal life. Conclusions: Survival five years after the procedure showed a beneficial outcome of the method used.", "keywords": [ "flow cytometry crossmatch", "HLA based on DNA", "pediatric renal transplantation" ], "content": "Background\n\nEnd stage renal disease (ESRD) is a relatively rare case in children with limited epidemiological data. In 2008, the prevalence of this disease was approximately 65 per million age-related populations in Australia, Canada, Malaysia and Western Europe.1,2 Dialysis, continuous ambulatory peritoneal dialysis (CAPD), and renal transplantation are three options for renal replacement therapy in ESRD. Renal transplantation is the preferred method of treatment for ESRD patients despite the risk of body rejection reactions as complications of the surgery, because of the least morbidity rate, the best survival rates, the best quality of life, and the best improvement in activities of daily living.3–5\n\nFlow cytometry crossmatch is a standard technique for evaluating the recipient’s and donor’s compatibility for renal transplants. The recipient’s serum, together with donor lymphocytes, is incubated and analyzed in a flow cytometer for the presence of IgG antibodies.6 Flow cytometry plays an important role in crossmatch before and after transplantation. When combined with human leukocyte antigen (HLA) immunophenotyping based on DNA, flow cytometry will have a more sensitive prediction for chronic graft rejection than complement-dependent cytotoxicity (CDC) crossmatching and is able to eliminate irrelevant antibodies (IgM).7\n\nFlow cytometry crossmatch can inform several things such as cytokine production in intracellular, analysis of phenotyping, and detailed information related to several T cells subset. By using tagged multimers with fluorescent, we could specifically calculate cellular antigen stimulation test (CAST). We can find MHC Class I or Class II monomers in multimers.8\n\nThe American Family Children's Hospital showed that the survival rates for post renal transplantation pediatric patients after 11 months, 1 year, and 3 years were 100%, 98%, and 97%, respectively. Meanwhile, graft survival is very dependent on the success of the renal transplantation. When compared with other therapeutic modalities, the prevalence of renal transplantation survival at 1 year was quite high at 97.9%, compared with CAPD (94.5%) and hemodialysis (87.3%). Over a longer period of time, the survival rate for post renal transplantation patients at 10 years was 86%, compared with CAPD (35%) and hemodialysis (33.8%).9\n\nIn our first experience of renal transplantation using flow cytometry screening and HLA immunophenotyping, we wanted to know the results at five years follow-up after transplantation.\n\n\nCase presentation\n\nWe present the case of a 20-year-old Javanese woman who underwent renal transplantation five years previously due to ESRD caused by bilateral parenchymatous renal disease. She is a student.\n\nShe had a history of hypertension since December 2014 and had undergone hemodialysis for three months. She came to the hospital with right and left lower back pain for the past year. She had high blood pressure of 140/90 mmHg, anemic conjunctivas and bilateral flank pain. Initial laboratory tests showed hemoglobin levels of 7.3 g/ dL, leukocytes 4750/ mm3, platelets of 174,000/mm3, blood urea nitrogen (BUN) 16.3 mg/ dL and serum creatinine 4.21 mg/dL. Anti-CMV IgG showed reactive results and a complete urine examination found albuminuria (2+). On radiological examination, chest radiograph and plain abdominal radiograph were within normal limits. Chest radiograph showed that heart in normal shape, size and position; lung in normal vascularization, bilateral hilum in good condition, and there were no infiltrate, cavity or nodul; there was no abnormality of aorta; trachea in normal position; costophrenic angle was sharply-pointed; bilateral hemidiaphragm in normal dome shaped; bones and soft tissues were normal. Abdominal radiograph showed that bilateral pre-peritoneal fat lines and psoas lines in normal condition; normal intestinal gas pattern; bones and soft tissues were normal. Abdominal ultrasound showed an increased echogenicity of bilateral renal cortex that could reflect a bilateral parenchymatous renal disease. Magnetic resonance angiography of the recipient showed bilateral decreased parenchymal thickness that reflected chronic parenchymatous renal diseases; ascites in the pelvic cavity; left and right common iliac arteries, internal iliac artery, external iliac artery were in normal vascularization and showed no thrombus or stenosis (Figure 1).\n\nPCR-SSP: polymerase chain reaction-based sequence specific primers; HLA: human leukocyte antigen; ABDR: AB serology and oligotyping for DR114.\n\nThe patient was subsequently diagnosed with stage V chronic kidney disease based on history of hypertension, high blood pressure (140/90 mmHg), anemic condition (HB: 7.3gr/dL) and elevated renal function test (urea 16.3 mg/dL and serum creatinine 4.21 mg/dL) and decreased glomerular filtration rate (GFR: 12,97 ml/menit/1,73m2). Differential diagnosis were diabetic nephropathy, chronic glomerulonephritis, nephrosclerosis, polycystic kidney disease, rapidly progressive glomerulonephritis but all other diagnosis had been excluded from the radiologic examination and no previous history of diabetes mellitus and no sign of hyperglycemia. She had undergone hemodialysis for three months. Flow cytometry and DNA-based HLA testing between the recipient and the donor was carried out to assess prediction of graft rejection prior to renal transplantation surgery. The HLA screening showed mismatch typing 3/6 and flow cytometry of T and B lymphocytes was negative.\n\nThe renal transplantation was performed in March 2015 by urologists from Malang (Malang’s Kidney Transplantation Team). Before the procedure, the patient had been optimized by hemodialysis prior surgery. Patient was under general anasthesia and in supine position. The transplantation procedure used the end-to-side anastomoses method on the donor renal artery with the right external iliac vein. The ureter from the donor kidney was neo-implanted into the recipient bladder using the Lich Gregoir technique, and a special double-J (DJ) stent No. 12 and retroperitoneal drain was placed. Doppler ultrasound examination was performed to assess the vascularization of the new kidney in the recipient's body. The result showed that the color flow didn’t reach the periphery, vascularization of the interlobular arteries was good and the surgical wound was closed. It turned out that there was tension in the fascia, reflected in abnormal resistive index and disproportion of larger size of the donor kidney to the smaller space in the recipient’s pelvic cavity and this resulted in the decreased blood flow to the kidneys seen in Doppler ultrasound evaluation. Then we decided to open the stitches and after that, ultrasound evaluation was carried out, which showed an improvement in blood flow. Finally, a mesh was placed on the recipient's fascia with the aim of reducing tension, then the wound was closed and reexamined with Doppler ultrasound, which showed that the color flow reached the periphery and the interlobular artery could be visualized without Doppler power (Figure 4).\n\nAfter the surgery, the recipient was directly admitted to the intensive care unit for five days with strict observation of fluid balance, electrolyte imbalance, blood pressure, and related laboratory parameters. After transplantation, the patient was given medication in the form of 500 mg Cellcept two times a day and a 5 mg Prograft two times a day as immunosuppressants. After two weeks of hospitalization, the patient showed good response to immunosuppressant drugs and showed no rejection reaction to transplantation. Then she was allowed to discharge from hospital at the early of April 2015 after two weeks in ward. She was asked to come back to urology clinic in the same hospital one month after discharged.\n\nA month later the patient came to urology clinic for evaluation of DJ-stent removal and it was done under local anasthesia by urologist from Malang. She was asked to come back at three months. Then she came again at three months with good results. When the patient returned for monitoring at the urology clinic at four months, the creatinine level had increased by 4.9 g/dl and the ultrasound results showed hydronephrosis grade II. It was then decided to redo surgery to find out the cause of hydronephrosis. The patient undergone hemodialysis prior surgery to optimize her condition. The surgery was done under general anesthesia and in lithotomy position. The patient underwent a ureterorenoscopic evaluation and right ureteral stenosis was found during the procedure. Then she underwent a ureterorenoscopy with a Holmium laser to dilate the ureteral orifice and a DJ stent was re-inserted into the patient. She was hospitalized for two days and discharged in the early of July 2015 after being two days in ward. She was asked to come back to urology clinic one month after discharged.\n\nA month later the patient came to urology clinic for the evaluation of DJ-stent removal. She was in a good condition and the ultrasound results showed that the size of transplanted renal was 10.1 cm × 5.6 cm with normal echocortex, hydronephrosis grade I, DJ-stent insitu, cortex-medulla border in normal condition, no sign of ureteral widening, and doppler parameters in normal condition (Figure 5).\n\nShe could do her usual activities of daily living three months after surgery, and during these three months she only did light activities in her house. After that she came routinely to urology clinic every month to monitor and evaluate her condition, laboratory and other supporting examination results.\n\nAfter five years post transplantation, the patient returned to outpatient care for routine appointments without any complaints. She also routinely consumed Herberser 100 mg once a day, Cellcept 500 mg twice daily, Prograf 0.5 mg once a day, Methyl Prednisolone 4 mg every two days, and Simvastatin 20 mg once a day. There were no abnormalities on physical examination. The latest laboratory test results showed a haemoglobin level of 11.9 g/dL, erythrocytes 4.790/uL, leukocytes 7,000/uL, hematocrit 38%, platelets 325,000/uL, iron 34 ug/dL, total iron binding capacity 301μg/dL, transferrin saturation 11%, albumin 4.3 g/dL, fasting blood glucose 93mg/dL, urea 33 mg/dL, creatinine 1.44 mg/dL, uric acid 5.9 mg/dL, sodium 140mmol/L, potassium 3.9 mmol/L, chloride 106 mmol/L, phosporous 3.3 mg/dL, and tacrolimus 3.8 ng/dL (tacrolimus levels in the last five years ranged from 3,5-7 ng/dl). Ultrasound examination of the patient revealed intrarenal vascularization, transplanted arteries and veins, and iliac arteries within normal limits (Figure 6). In these five years, the patient was routinely monitored at the urology clinic with serum creatinine levels between 1.5 to 2 mg/dL, routine hematology, albumin levels, serum urea, electrolytes, and tacrolimus levels within normal limits. She was able to carry out normal daily activities and is currently continuing her education at university.\n\n\nDiscussion\n\nRenal transplantation is the preferred treatment option in children with ESRD. Renal transplantation is currently considered the treatment option of ESRD for children because it is associated with better quality of life, productivity, and child growth, as well as longer survival than other modalities. There have been significant developments related to renal transplantation, including immunosuppressive medication therapy, grafting, and patient safety. These developments can contribute to many factors, including pre-transplant care, surgical development, and stronger immunosuppressant management.10,11\n\nHLA protein antigen on the cell surface is the key to differentiation of self-proteins from non-self-proteins. The two groups of MHC genes relevant to transplantation are class I and class II. HLA matching is a known predictor for the success of renal allograft. HLA typing is a crucial step in renal transplantation because recognition of foreign HLA by recipient T lymphocytes will trigger an immune response. Activation of T lymphocytes initiates a cascade of mediators that causes the immune system to fight allografts.12,13\n\nFlow cytometry crossmatch, as well as CDC crossmatching, also depends on donor lymphocytes which incubated with the receiving serum. Nevertheless, instead of a complement factor addition, a fluorescence coated second antibody was added which acted against IgG-DSA. These anti-IgG antibodies bind to the complex of donor Ag-DSA and allow the flow cytometer to detect. The result of reading is semi-quantitative and more objective because these antibodies instead of quantifying cell death visually, use a channel shift over the baseline. Flow cytometry crossmatch is more sensitive than CDC crossmatching because it does not depend on activity of complement. Negative value of flow cytometry crossmatch excludes the possibility of immunologically significant DSA. Furthermore, flow cytometry crossmatch is also more specific than CDC crossmatch because it only detects IgG antibodies and does not detect IgM.14\n\nA study has shown that flow cytometry has become the most sensitive approach for detecting HLA alloantibodies.15 The anti-human globulin-supplement dependent cytotoxicity (AHG-CDC) method provides a higher antibody detection sensitivity than CDC method. However, this technique is limited because of its low lymphocyte viability. On the other hand, flow cytometry crossmatch does not require cell viability or depend on complement fixation, but can detect immunoglobulin molecules that bind to target cells using immunofluorescence. Thus, low lymphocyte viability did not affect the results of flow cytometry crossmatch. In addition, flow cytometry crossmatch can detect not only complement-binding antibodies but also immunoglobulins that correct incompleteness. Therefore, flow cytometry crossmatch is more sensitive than CDC and AHG-CDC. Previous reports on a population in Thailand found that flow cytometry crossmatch was 8-32 times and 4-16 times more sensitive than CDC and AHG-CDC. They also found 28.9% of flow cytometry crossmatch to be positive in patients with negative CDC.16\n\nThe strengths in this case were:\n\n1. This case was the first five year follow up of pediatric renal transplantation using flow cytometry crossmatch and HLA immunophenotyping based on DNA for screening test in our hospital. So that we could know more how this procedure bring good hope for the patient and the donor, also how was the survival rate after five years, whether it followed the theory or not.\n\n2. A pilot project for other patient with the same procedure which had underwent procedure after this case to be followed and monitored for the survival rate.\n\nThe limitations in this case were:\n\n1. Only one patient had been followed up in this case report, leading to lack of data and needed more considerations and more patients to conclude the five-year survival rate.\n\n2. We could not determine the causality of the related factors due to descriptive study.\n\n3. Possibility of publication bias and overinterpretation due to only single case to be followed up and fortunately in this case showed good result.\n\nIn this case, the donor-recipient relationship is mother-daughter. The recipient was a 15-year-old girl with complaints of recurring low back pain since one year previously and had been diagnosed with hypertension and stage V chronic kidney disease with hemodialysis therapy two to three times a week for three months. Meanwhile, the donor was a 42-year-old recipient mother without a history of surgery or being hospitalized before. The HLA screening showed mismatch typing 3/6 and flow cytometry of T and B lymphocytes was negative. The renal transplantation was then carried out in March 2015.\n\nIt has been observed that patients with a greater risk of typing compatibility have a lower risk of acute rejection reactions as well as a better likelihood of transplant resistance. During this time, HLA typing was used with serology and then gradually adopted the molecular DNA method.17 Most of the tissue typing laboratories started HLA typing with serology and then gradually adopted the molecular method, first for class II and then for class I. In this case, flow cytometry crossmatch and HLA typing based on DNA were used to predict graft rejection between the recipient and donor.18\n\nIn a period of five years, this case showed good results with survival in a pediatric renal transplantation where the patient could continue normal activities and study at university. The patient's serum creatinine results ranged from 1.5-2 mg/dl and tacrolimus levels were also in the normal range between 3.5-7 ng/dl in these five years. Renal transplantation is thought to be more cost-effective than other renal replacement therapies such as lifelong hemodialysis, although direct economic analysis for renal transplantation in Indonesia has not been published.\n\nA brief perspective from the patient about the procedure and the follow up would be a challenging part for this case. The patient told us how grateful she was to have a brand-new life with a new fresh kidney thus she could do anything which she could not do in a limited life before. She felt free to resume usual activity and the risk of graft rejection could be predicted and prevented by using the flow cytometry crossmatch and HLA-immunophenotyping based on DNA for screening test. Even though she had to undergo many parts of procedure, such as preoperative hemodialysis, kidney transplantation, redo surgery, DJ-stent removal, consumed many pills, came to urology clinic every month to get monitoring and evaluation of treatment and the upcoming follow ups, she never regretted being part of the procedure. She felt so grateful and thankful for the treatment she got from us. The best part of her perspective was this method brings the diseased patient into a new era of meaningful and independent life. She could do a jogging time, follow any meetings and activities in her university.\n\n\nConclusion\n\nThe optimum follow-up results in the patient in this case suggest that renal transplantation in children can provide hope of survival of both the patient and the donor, even if this procedure is performed in developing countries, using the flow cytometry crossmatch screening method along with DNA-based HLA immunophenotyping.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of their details and clinical images was obtained from the patient.\n\n\nEthics\n\nEthical approval for this case report was obtained from The Ethics Committee of Saiful Anwar General Hospital, Malang with approval number 400/006/CR/302/2020.", "appendix": "References\n\nBecherucci F, Roperto RM, Materassi M, et al.: Chronic kidney disease in children. Clin Kidney J. 2016; 9(4): 583–91. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHarambat J, van Stralen KJ, Kim JJ, et al.: Epidemiology of chronic kidney disease in children. Pediatr Nephrol. 2012; 27(3): 363–73. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJefferies MT, Gupta A, Proctor A: The management of acute testicular pain in children and adolescents. BMJ. 2015. PubMed Abstract \| Publisher Full Text\n\nLee SM, Huh JS, Baek M, et al.: Nationwide Epidemiological Study of Testicular Torsion. J Korean Med Sci. 2014; 29(12): 1684–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSrinivasan A, Cinman N, Feber KM, et al.: History and physical examination findings predictive of testicular torsion: an attempt to promote clinical diagnosis by house staff. J Pediatr Urol. 2011; 7(4): 470–474. PubMed Abstract \| Publisher Full Text\n\nDowning J: The Lymphocyte Crossmatch by Flow Cytometry for Kidney Transplantation. In: Christiansen F, Tait B (eds) Immunogenetics. Methods in Molecular Biology (Methods and Protocols), vol 882. Totowa, NJ: Humana Press; 2012.\n\nHaarberg KM, Tambur AR: Detection of donor-specific antibodies in kidney transplantation. Br. Med. Bull. 2014; 110(1). PubMed Abstract \| Publisher Full Text\n\nMaguire O, Tario J, Shanahan T, et al.: Flow Cytometry and Solid Organ Transplantation: A Perfect Match. Immunol Invest. 2014; 43(8): 756–74. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAmerican Family Children’s Hospital: Dialysis and Transplant: Patient Survival Statistics.Reference Source. Last updated: 11/13/2015. Accessed 21 May 2020.\n\nSmith JM, Dharnidharka VR: Progress in pediatric kidney transplantation. Open Urol Nephrol J .2014; 7(1). PubMed Abstract \| Publisher Full Text\n\nSaeed B: Pediatric Renal Transplantation. Int J Organ Transplant Med. 2012; 3(2): 62–73. PubMed Abstract \| Free Full Text\n\nFernandez H: Application and interpretation of histocompatibility data in pediatric kidney transplantation. Curr Opin Organ Transplant. 2017; 22(4): 426–32. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlthaf M, El KM, Jin J, et al.: Human leukocyte antigen typing and crossmatch: A comprehensive review. World J Transplant. 2017; 7(6): 339–48. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGunawansa N, Roshni R, Ajay S, et al.: Crossmatch Strategies in Renal Transplantation: A Practical Guide for the Practicing Clinician. J Transplant Surgery. 2017; 1(1). Publisher Full Text\n\nHenel G, Schmitz JL: Basic theory and clinical applications of flow cytometry. Lab Med. 2007; 38(7): 428–36. Publisher Full Text\n\nKupatawintu P, Tatawatorn A, Premasathian N, et al.: Association between flow cytometric crossmatching and graft survival in Thai cadaveric-donor kidney transplantation. Asian Pac J Allergy Immunol. 2016; 34(1): 86–93. PubMed Abstract \| Publisher Full Text\n\nKoktathong K, Vejbaesya S, Bejrachandra S, et al.: Flow cytometric crossmatch for kidney transplantation. J Med Assoc Thai. 2005; 88(6): 769–774. PubMed Abstract\n\nPutra REA, Besut D: Pediatric renal transplantation using flow cytometry crossmatch and HLA immunophenotyping based on DNA for screening: a case report. Pan Afr. Med. J. 2018. Publisher Full Text\n\nMeiyanto JD, Besut D, Kurnia PS: A five-year follow-up after pediatric renal transplantation using flow cytometry crossmatch and HLA-immunophenotyping based on DNA for screening test. Abstract. Int. J. Urol. 2020; 27: 151. Publisher Full Text" }	[ { "id": "84926", "date": "09 Jun 2021", "name": "Ali Ersin Zumrutbas", "expertise": [ "Reviewer Expertise Urology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis case report was the first five-year follow-up of pediatric renal transplantation using flow cytometry crossmatch and HLA immunophenotyping based on DNA as a screening test in a local hospital. I would like to congratulate the authors for this case report including a comprehensive review and sufficient images. Minor grammatical and syntax errors should be corrected.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "86508", "date": "26 Jul 2021", "name": "Nur Rasyid", "expertise": [ "Reviewer Expertise Urology", "Kidney Transplantation", "Kidney Stone", "Andro-Urology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors presented their case report on a five-year follow-up after pediatric renal transplantation using flow cytometry crossmatch and HLA immunophenotyping based on DNA for screening test. This is a good case report, however some issues needed to be clarified further:\nAbstract:\nBackground: \"The survival rate at one, five and ten years has been shown to be 99%, 97% and 96%...\". Please elaborate on what survival rate these numbers are for. The previous sentences were about flow cytometry combined with HLA immunophenotyping.\n\nCase presentation: \"...and she could resume normal life\". Please use different vocabulary to define her quality of life post-transplantation.\n\nConclusion: \"Survival five years after the procedure showed a beneficial outcome of the method used.\" Please elaborate further on the word \"beneficial\" to objective parameters that are evaluated during this five-year follow-up.\n\nBackground:\nPlease elaborate further on renal transplantation conditions in Indonesia. In general, the source of the donor in kidney transplants can be from cadaveric or living donors. In Europe and Western countries, cadaveric donors are more prevalent than living donors. This is interesting as in Indonesia there are no reports on cadaveric donors since the regulation of using cadaveric donors is still in progress. In this case report, the donor is from her mother (living donor). In my opinion, it would be nice if the authors mention this condition also in the Background.\n\nIt would be better if you elaborate on why this is the first time your transplantation center used flow cytometry and HLA immunophenotyping. Is there a limitation of the healthcare facility or is it a procedure cost problem?\n\nParagraph 4, Line 3-4: \"...the prevalence of renal transplantation survival\". It would be better if you omit the words \"the prevalence of\".\n\nPlease include a sentence/paragraph explaining the optimal expected result for flow cytometry crossmatch combined with HLA immunophenotyping based on DNA\n\nCase presentation:\nParagraph 2, Line 1: Please change the first sentence in your second paragraph to describe the condition when your team first met her, such as \"She came to the hospital with right and left lower back pain for the past year\".\n\nParagraph 2, Line 6: Please summarize the chest radiography expertise to only the important aspects. Normal values that are unrelated to renal transplantation can be omitted.\n\nParagraph 2, Line 8: Misspelling of \"nodul\", It is supposed to be \"nodule\".\n\nParagraph 3. Line 1-3: There was no need to re-mention laboratory values that support your diagnosis. You have previously mentioned those in Paragraph 2, except for GFR.\n\nParagraph 4, Line 1: You don't need to mention \"urologists from Malang...\", as it was already described in your author and affiliation list.\n\nParagraph 4, Line 2: \"The patient had been optimized by hemodialysis prior surgery...\" should be \"...prior to surgery\". Please change \"optimize\" to another word more suitable to refer to a patient. For example: \"pre-treated\". Furthermore, please further elaborate on the details of the pre-operative hemodialysis in your case.\n\nParagraph 5, Line 3: It is more appropriate not to use the drug brand name in this case report. Instead, you can use the drug's generic name.\n\nParagraph 6, Line 2: \"...by urologist from Malang\" is not necessary, as you have mentioned the author's affiliation beforehand.\n\nParagraph 6, Line 3-4: \"...creatinine level had increased by 4.9 g/dL\". Please clarify whether the creatinine level rises to 4.9 g/dL or rises by 4.9 g/dL. Furthermore, the typical measurement unit for creatinine level is mg/dL. In your article, it was written in g/dL. Is this accurate or is it supposed to be mg/dL? Additionally, please use capital letters when typing \"liter\" in dL, instead of \"dl\" written in your article.\n\nParagraph 6: It would be great if you could provide us an endourology picture from the Holmium laser URS procedure.\n\nParagraph 9, Line 2: Please use the drug's generic name instead of its brand name.\n\nParagraph 9, Line 6: Previously, in Paragraph 2, you have used the term \"blood urea nitrogen (BUN)\", however, in this Paragraph you use the term \"urea\". It would be better to only use one term to maintain congruence.\n\nWhile this paper focuses on the transplant recipient, it would be very enlightening if you could also provide information regarding the donor after five years of kidney donation. How is the donor's general wellbeing?\n\nDiscussion:\nParagraph 1, Line 3: Please revise “longer survival” to “better/longer life expectancy”.\n\nParagraphs 2-3 could be further simplified or moved to the Background section to explain more about typing compatibility and its methods of examination.\n\nParagraph 5, Line 3: Please revise “42-year-old recipient mother” to “the donor was a 42-year-old female, who was the recipient’s mother…” to avoid misunderstanding.\n\nParagraph 6, Line 1: please revise “greater risk” to “greater/better match” to avoid misunderstanding.\n\nParagraph 6: It would be enlightening for the readers if ideal HLA typing compatibility and flow cytometry results for kidney transplantation from other studies are mentioned.\n\nParagraph 7: Please mention other studies that include yearly follow-up cases for kidney transplantation recipients to compare your result with other studies, such as QoL, laboratory results, and kidney function.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "94891", "date": "22 Sep 2021", "name": "Nicolas Guillaume", "expertise": [ "Reviewer Expertise Immunogenetics", "transplantation (kidney", "hematopoietic stem cells)" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors presented their first experience of renal transplantation using flow cytometry screening and HLA immunophenotyping. The authors cannot perform anti-HLA antibodies assays (screening and identification by CDC or Luminex assay). We have no information about the sensitivity of their flow cytometry crossmatch assay (the link between positivity and antibodies MFI by Luminex). They do not show at least a determination of antibodies screening by CDC. However, it is very unusual to publish this type of transplantation without antibodies data to perform at least a virtual crossmatch before flow cytometry crossmatch. In my opinion, this article cannot be indexed.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? No\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? No", "responses": [] }, { "id": "94677", "date": "05 Oct 2021", "name": "Shigeo Horie", "expertise": [ "Reviewer Expertise Urology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors reported the successful case of pediatric renal transplant after the first five-year follow-up. This was the first case using flow cytometry crossmatch and HLA immunophenotyping based on DNA for screening test in their institutions. They did a meticulous description of the case including surgical trouble, which will be quite useful for the readers. The background is well described. This case was well examined and the outcome of the transplant was excellent, reporting a good course of pediatric transplant.\nThe methodological description of crossmatch was not well described. This is the only drawback of this paper. The authors should elaborate more on the detailed description of the method of flow cytometry crossmatch.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-352
https://f1000research.com/articles/10-349/v1	06 May 21	{ "type": "Method Article", "title": "Validity and reliability of method used to analyse hair cortisol concentration", "authors": [ "Jordan Reid", "Katy Parker", "Lydia Clemens", "Matt Bristow", "Jordan Reid", "Katy Parker", "Lydia Clemens" ], "abstract": "Hair cortisol analysis is a method of analysing the stress hormone cortisol that offers great potential for helping researchers understand the long-term impact of stress and distress on the body. Hair analysis not only provides an excellent method of studying the average production of cortisol over weeks and months, but also the potential to understand cortisol levels several months before the hair was collected. Whilst research with hair samples for cortisol analysis is a fast-developing field, there has been less analysis of the methods used to analyse hair cortisol. We report two studies where the novel hair analysis method developed at the Anglia Ruskin university (ARU) Biomarker Laboratory was tested for reliability and validity. In study 1, 32 participants provided hair samples for an examination of the reproducibility of the hair cortisol analysis method. In study 2, 53 participants provided a hair sample cut from the scalp, and the methanol that the cortisol was extracted into was split between two tubes and assayed at two different laboratories with different methods (ELISA, LC-MS/MS). Overall, the results demonstrate that the methods developed to analyse hair cortisol in the ARU Biomarker Laboratory were both reliable and valid. The discussion considers further avenues for research and optimisation of the methodology.", "keywords": [ "HCC", "Hair Cortisol Concentration", "Cortisol" ], "content": "Introduction\n\nCortisol is a hormone secreted by the zona fasciculata of the adrenal cortex which binds to the glucocorticoid receptor (GR) found in almost every vertebrate cell. The ubiquity of the GR means that cortisol’s effects across the body are diverse and include metabolic (increased blood sugar production), immunological (such as reduced resistance to certain infections), circulatory (acute elevation of blood pressure), renal and central nervous system (such as altered mood and reduced libido) effects (Becker, 2001). Cortisol secretion follows a now well-defined circadian rhythm (Clow et al., 2004; Hucklebridge et al., 2005) and cortisol secretion can also be substantially increased in response to psychological or physiological stress (Dickerson & Kemeny, 2004). Cortisol’s wide influence over cellular function means that stress induced changes in cortisol have the potential for substantial changes in physiology, behaviour and health.\n\nResearch exploring psychosocial variables in relation to cortisol secretion have predominantly measured cortisol in saliva, blood or urine samples, which allow for the measurement of near-current levels of cortisol secretion (saliva and blood) and recent levels (urine). Whilst such research has proved hugely useful, not all research questions seek to explore near-current or recent cortisol output. Researchers who want to explore how personality, traumatic events, or mental health conditions, for example, impact on cortisol secretion and subsequent health may well find a measure of longer-term cortisol secretion to be very useful. The assessment of average cortisol activity over weeks and months is challenging, with the traditional measures from serum, saliva or urine as it requires sampling over many days with multiple samples a day to capture the full complexity of the circadian variation. In addition, the traditional measures of cortisol can only be used prospectively, whilst analysis of hair for cortisol offers the tantalising possibility of retrospective cortisol measurement. For researchers wanting to understand cortisol secretion over longer time periods, with the potential to measure cortisol secretion that occurred before the research had even started, the analysis of hair samples may well provide the solution.\n\nAnalysis of hair samples for measuring cortisol levels was first reported in the behavioural literature in 2004 (Raul et al., 2004) and since then there has been a rapid increase of research using measures of hair cortisol to explore stress and or the reaction of the body to challenge in some form. A recent meta-analysis (Stalder et al., 2017) identified 72 papers exploring some aspect of chronic stress with hair cortisol. There is a wide variety of research being undertaken. For example, higher hair cortisol in comparison to control groups has been found in unemployed individuals (Dettenborn et al., 2010) shift workers (Manenschijn et al., 2011), endurance athletes (Skoluda et al., 2012), chronic pain patients (Van Uum et al., 2008), alcoholics in withdrawal (Stalder et al., 2010), undergraduates students during term time (Stetler & Guinn, 2020), dementia caregivers (Stalder et al., 2014), people with post-traumatic stress disorder (Steudte et al., 2011) and in children with early life adversity (Aas et al., 2019; Bhopal et al., 2019).\n\nUnderpinning the burgeoning use of hair cortisol in psychological research is the methodology used to determine the concentration of hair cortisol found in the hair sample. There is no single standardised approach at present for the measurement of hair cortisol but laboratories largely use a similar sequence of steps: the hair is washed to remove contaminants from the surface of the hair, the washed hair is dried, the sample is then cut or ground to increase the surface area of the hair available to extract cortisol from, the cortisol is extracted into a solvent (usually methanol over a period of time), the methanol and hair are separated, and the methanol is evaporated to dryness and analysed for cortisol concentration. The choice of assay method varies considerably but the commonest methods used are liquid chromatography with tandem mass spectrometry (LC-MS/MS) or an antibody-based assay, commonly enzyme-linked immunosorbent assay (ELISA).\n\nThe methodology developed by our laboratory was intended to be a scalable method of hair analysis that could be undertaken for large numbers of hair samples and had the minimum number of steps to reduce the chance of error and reduce costs. Our method reduces the number of steps by not transferring the sample to another container; it is kept in the same tube from initial weighing through to the end of the methanol incubation. The procedure is also very scalable. The ‘rate-limiting’ step for our hair analysis procedure is the grinding of the hair and this has been partially automated and is highly scalable. Final quantification of cortisol is performed via an ELISA assay, an assay which is rapid and cost effective.\n\nIn this paper we describe the methodology developed by the Anglia Ruskin university (ARU) Biomarker Laboratory and two studies that evaluate how reproducible our method is and the extent to which our assay results relate to LC-MS/MS analysis performed at another laboratory.\n\n\nAims\n\nThis study aimed to test the reproducibility of the hair cortisol assay and to develop a protocol that allowed the laboratory team to generate a number of aliquots of hair that had the same or very similar levels of hair cortisol.\n\nStudy 1 demonstrated that the hair assay procedure developed at the ARU Biomarker laboratory was highly reliable, but it did not demonstrate that the method creates valid results. To test this key hypothesis, we split the methanol, into which the hair cortisol extracts, into two tubes rather than the standard procedure of using one tube. One of the pair of tubes was analysed for cortisol via LC-MS/MS at Dresden Labservice GmbH, a world leader in hair cortisol testing, and one via the ELISA in the ARU Biomarker Laboratory, as per study 1.\n\n\nMethods\n\nParticipants\n\nA total of 30 male and two female participants aged between 18 and 55 years old were recruited to participate in this study using an opportunity sample of people having their hair cut in a specific barbers in Cambridge, UK. Hair samples were collected over multiple visits to barbers over eight days. The Participants were included if their hair was 3 cm or less in length and they were Caucasian. There were no other inclusion or exclusion criteria. Recruitment was stopped once 32 samples had been recruited as this number would enable us to fit all of the replicate A and B samples on the same ELISA plate, thus removing any inter assay variability from the analysis. In addition, 32 samples would give us substantial power (.99) to detect a correlation greater than r = .65, should such a relationship exist between the two variables. Given that replicates A and B should correlate very highly this was considered to be an adequate sample size.\n\nDesign\n\nThere were three linked experiments in study 1.\n\n(1) Reproducibility: From each participant, two aliquots were analysed for hair cortisol with the aim of testing the reproducibility of the assay.\n\n(2) Incubation times: Hair samples from each participant were tested using the hair cortisol method after 3, 21, 24, 28 and 48 hours of methanol incubation. This was intended as an initial test to identify where the hair cortisol concentration (HCC) plateaued during the methanol incubation.\n\n(3) Grinding media: Hair samples from each participant were analysed using the standard hair cortisol methodology using either three or five ceramic grinding balls.\n\nHair retrieval protocol\n\nHair was collected from volunteers from people who were having their hair cut at local barbers. Participants were given information about the research after arrival at the barber shop and those who agreed to participate were asked to complete a written informed consent form and a brief hair questionnaire, which asked for specific detail of their hair treatment, for example, what styling products were used (this data is not reported further).\n\nOnce the barber was in place and ready to begin shaving, one of the authors (JR) held a small, rectangular plastic container approximately 15 centimetres below the barber’s clippers, to collect the shaved hair from temporal and occipital regions of the participant’s head. The collected hair was stored in a tin foil’envelope’, so the hair was stored securely but not hermetically sealed, and the samples were returned to the ARU Biomarker laboratory for storage at room temperature.\n\nTo create aliquots of the hair samples for analysis the participants’ samples were mixed using two clean tweezers for five minutes to ensure a thorough mixing of the hair. The aliquots were weighed using a precision balance. The hair from each participant was aliquoted in 25 mg amounts into 4.5 ml PPCO vials that are used from the weighing stage through to the methanol incubation.\n\nHair analysis protocol\n\nHair was analysed using the method reported by Parker & Bristow (2020). 25mg samples of hair in the 4.5-ml PPCO vials were each washed twice in 2 ml of isopropanol to remove external contaminants. The isopropanol was then removed from the vial and the hair allowed to dry in a microbiological safety cabinet for a minimum of 48 hours until fully dry. Once fully dry, five ceramic balls (Lysing Matrix M, MP Biomedicals, LLC) were added to each tube and the hair samples ground to a powder using Fast Prep-241 (MP Biomedicals, LLC). To extract cortisol, we added 2 ml of methanol to each sample and incubated the samples for 24 hours at room temperature rotating the samples constantly.\n\nThe hair, methanol and ceramic balls were decanted into a polypropylene tube (product code: 51.1534, Sarstedt AG & Co, Germany) and centrifuged at 1500 RCF to separate the ceramic balls from the rest of the mixture. The tube was then centrifuged at 3000 RCF to separate the ground hair and methanol, and 1.4 ml of the clear methanol supernatant was decanted into a 2-ml polypropylene microtube (product code: 525-0539, VWR). The supernatant microtubes were then centrifuged to dryness in vacuum centrifuge (Scan Speed 40, Labgene) at 37°C and 1700 RPM for three hours, and then frozen at -80°C until required for the cortisol ELISA. Cortisol levels were determined using a commercially available competitive ELISA (product code: #1-3002; Salimetrics, US).\n\nSamples were thawed and reconstituted with Salimetrics’ cortisol assay diluent and the samples were then assayed in accordance with the manufacturer’s protocol. A Salimetrics high and low control sample run in each ELISA plate run. The 32 samples for HCC replicates A & B were run in singlet to ensure that they were all run on the same assay plate initially. All other samples from the study were run in duplicate. Samples were re-run in duplicate at a higher dilution if the ELISA cortisol concentration was higher than 3 μg.dl−1. Replicate repeats were accepted as cortisol concentration coefficient of variation for the duplicates was less than 15% (unless the absolute difference between repeats was within 0.03 μg.dl−1). The samples were run over 9 plates with an inter assay coefficient of variation for the high control of 3.62% and 3.08% for the low control. The intra assay coefficient of variance was 3.77%. The results were expressed as the picograms of cortisol per milligram of hair.\n\nParticipants\n\nThe participants in study two were separate from those in study one. Staff and students in the ARU Department of Psychology were invited to participate in this study by email and advertisements in lectures and were offered £2.50 or 0.5 research credit for participation. We expected to find a strong correlation between ELISA and LC-MS/MS and the power calculation study 1 remained the same that we wanted a minimum of 32 participants to test the correlation between ELISA and LC-MS/MS and that this would give us substantial power to detect a correlation of greater than 0.65, should that exist. Fifty-three people (eight males, 45 females and one person who declined to answer) with a mean age of 20.98 years (SD: 4.15) provided a hair sample for analysis. The only inclusion criteria was having hair at least 1cm long. One person accidentally provided two separate hair samples one week apart, so the first hair sample provided was used in the analysis and the other excluded from analysis. All participants gave written informed consent to participate.\n\nDesign and analysis\n\nA hair sample (see hair collection, below) was taken from each participant along with a brief demographic questionnaire that asked questions about age, gender and a range of questions about the participants height, weight and hair washing methods that are not reported further. The ARU Biomarker Laboratory extracted the cortisol from the first 1 cm of hair from the scalp into methanol using the methods detailed in study 1. The methanol from each sample was then split equally into two polypropylene tubes (0.7 ml per tube) and dried down using the standard protocol. The tubes were then stored at -80°C. One set of tubes was then sent to Dresden Labservice GmbH for analysis via LC-MS/MS (see Gao et al., 2013) and the other set was analysed via ELISA at the ARU Biomarker Laboratory using the same methods detailed in study 1. All sample were analysed in duplicate. The samples were run over 2 plates with an inter assay coefficient of variation for the high control of 9.75% and 10.16% for the low control. The intra assay coefficient of variance was 9.64%. The results were expressed as the picograms of cortisol per milligram of hair.\n\nHair collection\n\nHair samples were collected from each participant from the posterior vertex of the scalp as this area has the lowest coefficient of variation compared to hair sampled from other areas of the scalp (Sauvé et al., 2007). The hair sample size was approximately a five mm diameter of hair in total and the samples were taken from two different locations of the posterior vertex to reduce the visible signs that hair had been cut. The hair was tied with linen thread as close the scalp as possible and the hair was then cut as close to the scalp as possible using clean scissors and stored in a tin foil envelope at room temperature.\n\nThe research was approved by the Faculty of Science & Engineering Faculty Research Ethics Panel at Anglia Ruskin University (Study 1 ethics number FST/FREP/18/805; Study 2 ethics identifier: FST/FREP/13/378). All participants provided signed informed consent before participation.\n\nAnalysis was conducted using Jamovi 1.63 (The Jamovi Project, 2020) and IBM SPSS version 26 (IBM SPSS, 2019) with p < .05 considered as a significant difference. Power Analysis used GPower 3 (Faul et al., 2007). For all HCC variables in study 1 there were three extreme outliers (see Figure 1A) that were more than three times higher than the interquartile range from Tukey’s hinge at the 75th centile, and a further two points that were outliers for most variables. (Figure 1B shows the relationship between replicates with the five outliers removed.) In addition, the hair cortisol variables show a significant positive skew and kurtosis. The HCC data in Study 2 also contained a number of outliers with a positive skew and significant deviation from normal distribution. Table 1 shows descriptive statistics for the full datasets of both study one and two.\n\nMany researchers working with hair cortisol use log transformations to reduce the skew seen with hair cortisol and to reduce the impact of high outlying values that are often found. We have not used log transformations in our analysis, partly because of theoretical objections that researchers have raised to log transformations (Feng et al., 2014) and on a practical level because log transformations did not remove the distribution problems observed in our data. We opted instead to use non-parametric analysis and to provide supplemental parametric analysis where it may be helpful to compare with previous research. Where parametric analysis is used it is noted along with the outliers removed.\n\nIn both studies Bland-Altman plots (Bland & Altman, 1986) were used to analyse the agreement between the methods rather than the association (assessed via correlation). When Miller, Plessow, Rauh, Gröschl, & Kirschbaum (2013) compared the ELISA and LC-MS/MS results with salivary analysis, they reported high correlations between the two methods but that all ELISA results were higher than the LC-MS/MS method (ranging between 27.9% and 259.3% higher depending on the ELISA assay used). It is therefore not our expectation that the LC-MS/MS and ELISA will agree in absolute terms but rather than the difference between the two methods will be consistent over the range measured.\n\nSpearman’s Rho non-parametric correlation was used to measure the association between the HCC replicates A and B (Study 1) and ELISA and LC-MS/MS HCC from the same hair cortisol extract (Study 2) and Friedman’s non-parametric test for repeated measures used to test for a difference between the hair cortisol replicates A & B in study 1 and between ELISA and LC-MS/MS analysis in study 2. Pearson’s Product Moment correlation and Student’s t-test have been used to provide a parametric version of the above analysis to allow comparison with other research. Friedman’s repeated measures non-parametric test was used to test whether the HCC different across the five different incubation times for hair and between samples ground with 3 ceramic balls or 5 ceramic balls. Where there were more than two levels Durbin-Conover pairwise tests used to identify the location of any differences.\n\n\nResults\n\nThere was a very significant correlation between the two replicates of hair cortisol using the full dataset (rs = .967, N = 32, p < .001; r = .995, N = 32, p < .001, see Figure 1A) (Reid, Parker & Bristow, 2020). Friedman’s test revealed no significant difference between the two replicates (χ2 = .50, df = 1, p = ns). As noted above, parametric analysis on this dataset is unreliable given the distribution of the data but to allow a better comparison with other published work we have included some parametric analysis. With the five outliers the Shapiro-Wilk test for normality of data remains significant indicating that the data is not normally distributed. The Pearson’s product moment correlation between the replicates remains highly significant (r = .926, N = 27, p < .001; rs = .945, N = 27, p < .001) and there was no significant difference in the means for replicate A (7.78 mg.pg-1; SD = 3.27) and replicate B (7.46 pg.mg-1, SD = 3.06)(t = 1.35, df = 26, p < .190). The Bland-Altman plot (see Figure 2 below) suggests a good agreement across the range of HCC means though two points fall on or outside the ± 1.96 lines of agreement.\n\nFive outliers have been removed from the data. The top and bottom lines indicate + or – 1.96 standard deviations from the replicate differences.\n\nThe Friedman non-parametric repeated measures test indicated a significant difference between five incubation times (3, 21, 24 and 48 hours; χ2 = 10.9, df = 4, p = .027. Durbin-Conover pairwise comparisons showed that the 24 h HCC were significantly lower than the other levels but there were no other significant differences. The Friedman n test revealed no significant difference between using three or five ceramic balls (χ2 = 1.00, df = 1, p = ns) to grind the hair samples.\n\nThe LC-MS/MS hair cortisol concentrations were significantly lower than the ELISA concentration (χ2 = 32.7, df = 1, p < .001; see Table 1) but there was a very strong correlation between the LC-MS/MS and ELISA hair concentrations (rs = .905, n = 53, p < .001; r = .911, n = 53, p < .001) (Bristow, Clemens & Parker, 2020). The scatterplot showed a strong linear relationship with one discrepant value (see Figure 3). The Bland-Altman plot (see Figure 4) also clearly indicated one value with a highly anomalous result but otherwise the values fell within the boundaries.\n\nThe top and bottom lines indicate + or – 1.96 standard deviations from the replicate differences.\n\n\nDiscussion\n\nOur initial experiment in Study 1 examined how well two aliquots of hair from the same person yielded the same hair cortisol concentration. This experiment tested two interlinked research questions: 1) were the aliquots of hair that had been created very similar for hair cortisol levels? And 2) were our assay results reproducible? We found a very high correlation between the two aliquots of hair and this supports both questions with no evidence of difference between hair cortisol levels found in the two aliquots. In addition, the average coefficient of variance for the HCC of the two replicates was 8.51% (SD: 6.2%), which again shows a close agreement between the replicates. Correlations are not the ideal way to measure agreement between two laboratory measures, and the Bland-Altman plot can be of more use (Grouven et al., 2007). The Bland-Altman plot (see Figure 2) shows that all bar two of the datapoints were within the ± 1.96 limits of agreement and there is no pattern in the plotted data that would suggest any systematic bias. There are two points that lie outside the limits of agreement, one high and one low, and these both came from samples towards the top of the range of concentrations reported.\n\nOur results showed that the error was being kept to a low level and the results were reproducible. This supports both our research questions. We can be confident that the aliquots of hair created have similar cortisol levels in each aliquot and that our assay is providing a reproducible result with the current protocol.\n\nHaving established that the aliquots were very similar in hair cortisol levels and that our assay showed high reproducibility, we wanted to explore whether the incubation times we are currently using are optimal and whether the grind quality could be reduced without impacting performance.\n\nThe incubation test was intended to be the first of a series of tests to understand the time of peak extraction of cortisol and understand the variability of extraction around the peak extraction point. We might have expected that incubating for three hours would have extracted less cortisol because of the relatively short extraction time and the timings we chose for this first test were aimed at giving us more information around the 24-hour time, as this is the time that we currently use. It is of interest for us at what point the cortisol extraction into methanol peaks and plateaus. Though 24 hours provides a highly reproducible result, it may be that we can optimise the assay further with a longer incubation or that we can reduce the incubation times, which may allow for faster analysis of samples. Unexpectedly, we found that hair cortisol levels were stable over the incubation times except that the 24-hour incubation was slightly, but significantly, lower than the other incubation times. It is of interest that HCC shows no sign of increasing with incubation and then plateauing. Instead we have – save for a very small dip at 24 hours – the same level of extraction over the time. This means that the 24-hour incubation period is not necessary for maximal extraction and we could likely achieve the same results in three hours, or even shorter. It also likely means that the hair grind level could be reduced. We currently grind to a very fine grind which exposes a substantial surface area for methanol to extract cortisol from and appears to allow for a fast extraction time. Reducing the number of ceramic balls from five to three does visually reduce the fineness of the grind (unpublished observations from our laboratory) but we see no impact on the HCC extracted at 24 hours. Aside from reducing laboratory testing costs this suggests that research should look at reducing the degree of grinding and how this impacts on extraction of HCC. Grinding clearly damages the hair and it may be that we can achieve the same or better extraction of hair cortisol with less grinding.\n\nWe can find no reason for this slight fall in HCC at 24 hours. The samples were all assayed at the same period and the samples were randomly distributed over the ELISA plates used. There is always a minor variation between ELISA plates: had we had allocated all of the 24-hour incubation samples to one plate alone and the other conditions to different ELISA plates, this might have accounted for this difference, however this did not happen and the 24-hour samples were randomly allocated over plates. There was no difference in the hair weights for the various levels of incubation (F4,96 = 2.30, p = ns). Indeed, the hair weights were consistent with a range of 24.5 mg to 25 mg across all the conditions. There is no reason the samples would extract slightly less at 24 hours than they would at 21 hours or 28 hours (or indeed three hours or 48 hours) other than random chance or a small systematic error in the laboratory work on the 24-hour samples which may have been processed separately to the other incubation times. For example, the 24-hour aliquots of hair may be the ones that had, by chance, the slightly lower levels of hair cortisol in for that participant or that the researcher chose a different pipette to aspirate off the supernatant than selected for the other conditions, and whilst both pipettes are calibrated one may pipette slightly less than the other. In normal laboratory work, the small differences would not be observed, but given the nature of this experiment and the high reproducibility of the assay, an effect may be observed. The incubation test needs to be repeated in conjunction with varying grinds of hair to further optimise the assay but the slight drop at 24-hours does not create any issue for our standard assay protocol as all of our analysis is currently carried out with a 24-hour methanol incubation.\n\nStudy 1 supports the view that the hair cortisol assay provides highly reproducible results, something that is essential for any well-functioning assay. The other key aspect of any assay is whether the results obtained measure what they claim to measure.\n\nThe results from study 2 showed that ELISA method significantly over reported HCC with the median average for ELISA 2.57 pg.mg−1 (66.9%) higher than the median for LC-MS/MS. This inflation of the cortisol results is not trivial but is also not unexpected based on the previously mentioned finding from Miller et al. (2013) that LC-MS/MS measures of salivary cortisol where significantly lower than those found by ELISA. There was very good correlation between the LC-MS/MS and ELISA measurement of cortisol extracted from the hair samples in study 2 and is comparable to correlations reported between saliva and LC-MS/MS by Miller et al.\n\nThe Bland-Altman plot (see Figure 3) showed no clear systematic pattern in the differences with the caveat that as in study 1 the distribution was substantially positively skewed with most values falling at the lower end but with a tail of large values.\n\nThere is one very discrepant value in the results where LC-MS/MS recorded 19.48 pg.mg−1 compared with 7.44 pg.mg−1 from the ELISA method, odd since ELISA tended to over report hair cortisol in this study. Analysis via LC-MS/MS or ELISA is not perfect and there is always a small chance of an undetected technical issue, operator error or accidental contamination. This discrepant value may also, of course, reveal a potential confound which is causing one of the two assays types to give a false reading. We recorded a wide range of information about how people treated their hair, and it is perhaps noteworthy that the participant whose hair showed the discrepant values had dyed their hair the night before it was cut. Only one other participant in our study had dyed their hair so close to having their hair sample taken. This participant had accidentally been allowed to enrol in the study twice and had given a hair sample a week before they dyed their hair and then a second time on the day their hair was dyed. We had excluded this second sample from analysis as we could not have two samples from the same person. However, there is no indication from this excluded sample of any additional discrepancy between the ELISA measure (7.73 pg.mg−1) or the LC-MS/MS measure (4.39 pg.mg−1). This is a 75.9% inflation by the ELISA, which is not far from the average median inflation found in by the ELISA method in the study of 66.9%.\n\nBy splitting the extracted methanol between ELISA and LC-MS/MS we are explicitly testing the measurement of HCC in the extract rather than testing whether given samples of the same hair, both laboratories would arrive at the same result. Our results do show that with ELISA analysis of the extract our methodology produces a good correlation with LC-MS/MS analysis of the same extract. Testing the relationship between two different hair analysis methods from grinding through to analysis by ELISA or LC-MS/MS would be useful but any differences in the results could be due to the hair extraction methodology used in the other laboratory rather than the end assay used (ELISA vs LC-MS/MS).\n\nIn this article we report the methodology used by the ARU Biomarker Laboratory to analyse hair cortisol levels via ELISA and two studies used to test the reproducibility and validity of the assay. The assays show high reproducibility and high validity, and the data suggest the methodology is fit for purpose. The methods used in study 1 to create a series of aliquots of the same participant’s hair was successful in allowing us to have access to multiple samples of each person’s hair. This allows for a range of validation testing to be carried out on the same hair samples.\n\nThere is considerable scope for further optimisation work for the assay. The incubation times can be usefully analysed along with different degrees of grind from uncut hair through rough cut to fine powder to explore the lowest degree of grind that can produce reproducible results. Our initial development work (unpublished observations) suggested that 25 mg of hair provided reproducible results but that a range of around 10 mg to 100 mg of hair could be used to produce reproducible results. Outside these values there were concerns about the reproducibility. A more systematic evaluation of this could help researchers better understand the amount of hair required for reliable testing and the reliability of analyses conducted on samples outside of this range.\n\nThe research presented here confirms the hair analysis methodology developed at ARU Biomarker laboratory is both a reproducible and valid measure of hair cortisol. This allows for a scalable method for analysing cortisol levels from hair that can be undertaken for large numbers of hair samples while minimising the number of steps to reduce chance of error and costs. The findings evidence the reproducibility and validity of hair cortisol analysis as a measure of psychological and physiological stress. This type of work is essential in underpinning the application of hair cortisol measurement to increase our understanding of longer-term impacts of psychological stress on the human body.\n\n\nData availability\n\nFigshare: Hair cortisol concentration method validation study 1. https://doi.org/10.6084/m9.figshare.13352822.v3 (Reid, Parker & Bristow, 2020)\n\nThis project contains the following underlying data:\n\n• Study 1 hair validation Reid repository in.sav and.csv formats (The files contain the hair cortisol concentrations (HCC), the underlying ELISA measurement of cortisol (not corrected for hair weight) and hair weights for each condition in study 1 (replicate A & B, incubation over 3, 21, 24, 28 & 48 hours and a 3 ceramic ball grind condition))\n\n• Study 1 Reid detailed ELISA data.csv (This file contains the detailed ELISA (Enzyme Linked Immunosorbent Assay) records for all of the hair cortisol data. This includes replicate data for cortisol concentration, optical density (OD; 450nm) and difference optical density (620nm), the coefficient of variation for cortisol and difference optical density replicates and the sample dilution.)\n\nFigshare: Hair cortisol concentration method validation study 2. https://doi.org/10.6084/m9.figshare.13359695.v3 (Bristow, Clemens & Parker, 2020).\n\nThis project contains the following underlying data:\n\n• Study 2 ELISA LCMSMS Reid repositoryv2.sav\n\n• Study 2 LCMSMS Reid.csv (The.sav and.csv files contain the hair cortisol concentrations (HCC), the underlying ELISA measurement of cortisol (not corrected for hair weight) and hair weights for the samples analysed by both LC-MS/MS and ELISA. In addition, the files details which ELISA plate the samples were processed on and the order in which the aliquots of methanol went into the tubes (1 = ELISA tube first and 2 = LCMS/MS tube first). Age, BMI, height, weight, medication, hair colour and hair washing variables have been removed as they may allow identification of participants.)\n\n• Study 2 Reid ELISA data.csv (This file contains the detailed ELISA (Enzyme Linked Immunosorbent Assay) records for all of the hair cortisol data. This includes replicate data for cortisol concentration, optical density (OD; 450 nm) and difference optical density (620 nm), the coefficient of variation for cortisol and difference optical density replicates and the sample dilution. There is a sample number to identify which samples came from the same participant but this a randomised code and does not relate to the original participant numbers.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nAas M, Pizzagalli DA, Laskemoen JF, et al.: Elevated hair cortisol is associated with childhood maltreatment and cognitive impairment in schizophrenia and in bipolar disorders. Schizophr Res. 2019; 213: 65–71. PubMed Abstract \| Publisher Full Text\n\nBecker KL: Principles and Practice of Endocrinology and Metabolism. 3rd ed. Lippincott: Williams & Wilkins; 2001.\n\nBhopal S, Verma D, Roy R, et al.: The contribution of childhood adversity to cortisol measures of early life stress amongst infants in rural India: findings from the early life stress sub-study of the SPRING cluster randomised controlled trial (SPRING-ELS). Psychoneuroendocrinology. 2019; 107(November 2018): 241–250. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBristow M, Clemens L, Parker K: Hair cortisol concentration method validation study 2. figshare. Dataset. 2020. Publisher Full Text\n\nClow A, Thorn L, Evans P: The awakening cortisol response: methodological issues and significance. Stress (Amsterdam, Netherlands). 2004; 7(March): 29–37. PubMed Abstract \| Publisher Full Text\n\nDettenborn L, Tietze A, Bruckner F, et al.: Higher cortisol content in hair among long-term unemployed individuals compared to controls. Psychoneuroendocrinology. 2010; 35(9): 1404–1409. PubMed Abstract \| Publisher Full Text\n\nDickerson SS, Kemeny ME: Acute stressors and cortisol responses: a theoretical integration and synthesis of laboratory research. Psychol Bull. 2004; 130(3): 355–391. PubMed Abstract \| Publisher Full Text\n\nFeng C, Wang H, Lu N, et al.: Log-transformation and its implications for data analysis. Shanghai Arch Psychiatry. 2014; 26(2): 105. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFaul F, Erdfelder E, Lang AG, et al.: GPower 3: A flexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behav Res Methods. 2007; 39(2): 175–191. PubMed Abstract \| Publisher Full Text\n\nGao W, Stalder T, Foley P, et al.: Quantitative analysis of steroid hormones in human hair using a column-switching LC-APCI-MS/MS assay. J Chromatogr B Analyt Technol Biomed Life Sci. 2013. PubMed Abstract \| Publisher Full Text\n\nGrouven U, Bender R, Ziegler A, et al.: Comparing methods of measurement. Deutsche Medizinische Wochenschrift. 1946; 132Suppl(24), 69–73. PubMed Abstract \| Publisher Full Text\n\nHucklebridge F, Hussain T, Evans P, et al.: The diurnal patterns of the adrenal steroids cortisol and dehydroepiandrosterone (DHEA) in relation to awakening. Psychoneuroendocrinology. 2005; 30: 51–57. PubMed Abstract \| Publisher Full Text\n\nManenschijn L, Koper JW, Lamberts SW, et al.: Evaluation of a method to measure long term cortisol levels. Steroids. 2011; 76(10–11): 1032–1036. PubMed Abstract \| Publisher Full Text\n\nMiller R, Plessow F, Rauh M, et al.: Comparison of salivary cortisol as measured by different immunoassays and tandem mass spectrometry. Psychoneuroendocrinology. 2013; 38: 50–57. PubMed Abstract \| Publisher Full Text\n\nParker K, Bristow M: Hair cortisol analysis protocol. protocols.io 2020. Publisher Full Text\n\nRaul JS, Cirimele V, Ludes B, et al.: Detection of physiological concentrations of cortisol and cortisone in human hair. Clin Biochem. 2004; 37(12): 1105–1111. PubMed Abstract \| Publisher Full Text\n\nReid J, Parker K, Bristow M: Hair cortisol concentration method validation study 1. figshare. Dataset. 2020. Publisher Full Text\n\nSauvé B, Koren G, Walsh G, et al.: Measurement of cortisol in human hair as a biomarker of systemic exposure. Clin Invest Med. 2007; 30(5): 183–191. PubMed Abstract \| Publisher Full Text\n\nSkoluda N, Dettenborn L, Stalder T, et al.: Elevated hair cortisol concentrations in endurance athletes. Psychoneuroendocrinology. 2012; 37(5): 611–617. PubMed Abstract \| Publisher Full Text\n\nStalder T, Kirschbaum C, Heinze K, et al.: Use of hair cortisol analysis to detect hypercortisolism during active drinking phases in alcohol-dependent individuals. Biol Psychol. 2010; 85(3): 357–360. PubMed Abstract \| Publisher Full Text\n\nStalder T, Tietze A, Steudte S, et al.: Elevated hair cortisol levels in chronically stressed dementia caregivers. Psychoneuroendocrinology. 2014; 47(0): 26–30. PubMed Abstract \| Publisher Full Text\n\nStalder T, Steudte-Schmiedgen S, Alexander N, et al.: Stress-related and basic determinants of hair cortisol in humans: A meta-analysis. Psychoneuroendocrinology. 2017; 77: 261–274. PubMed Abstract \| Publisher Full Text\n\nStetler CA, Guinn V: Cumulative cortisol exposure increases during the academic term: Links to performance-related and social-evaluative stressors. Psychoneuroendocrinology. 2020; 114. PubMed Abstract \| Publisher Full Text\n\nSteudte S, Kolassa IT, Stalder T, et al.: Increased cortisol concentrations in hair of severely traumatized Ugandan individuals with PTSD. Psychoneuroendocrinology. 2011; 36(8): 1193–1200. PubMed Abstract \| Publisher Full Text\n\nVan Uum SH, Sauve B, Fraser LA, et al.: Elevated content of cortisol in hair of patients with severe chronic pain: a novel biomarker for stress. Stress. 2008; 11(6): 483–488. PubMed Abstract \| Publisher Full Text\n\n\nFootnotes\n\n1 The grinding settings are: 6 m/s for 30 seconds × 6 with 5 min cool down between grinds." }	[ { "id": "86211", "date": "20 Aug 2021", "name": "Yolanda B. de Rijke", "expertise": [ "Reviewer Expertise Endocrine disorders" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors described part of their analytical validation of theELISA (Salimetrics) method used in the ARU Biomarker Laboratory for hair cortisol. However, such a validation is mandatory for every accredited lab and does not involve any innovation in the method. Why didn't you provide information on the LLoQ and matrix interfence?\nThe scientific value of the paper is low. However, for the laboratories measuring (or would like to measure in future with the Salimetrics method) hair cortisol the information is still useful because information is provided on the number of ceramic balls and the extraction time. I have the following comments:\n1. Do the authors participate in the international round Robin to monitor their trueness of the method?\n2. Please provide a reference of the Dresden method on page 4 under study 2.\n3. Page 8, 1st paragraph: the authors referred to salivary analysis but hair is different from saliva and that difference should be emphasized.\n4. Page 7, Table 1. A lot of information is included but it is difficult to read without text of the paper and should be modified. - the SD is about 2-2.5 times the mean for the ELISA and about the same for study 2?\n5. Page 8, results: a. Figure 3 is informative and figure 1 can be deleted. b. Figures 1A and B have different axis-names? Legends of the figures are not sufficient to understand the figures, e.g. method, definition of replicate A and B\n6. Page 9, figure 4 is informative enough and figure 3 can be deleted.\n7. Page 10, 6th paragraph, last sentence is not right: \"...is comparable to correlations between saliva and LCMS/MS\". You compare material to a method and it should be modified. I believe results have been reported for saliva etc. Is it right that Miller also looked at the Salimetrics assay? If not, please state that.\n8. Page 11, 4th paragraph: You mentioned that a range of 10 to 100 mg of hair could be used. I do not agree that an amount higher than 40 mg is good for the assay because of the matrix interference and if you do not provide data on that range you should delete the entire sentence.\n9. In the LCMSMS Reid.csv file information on age and medication was not included on purpose. However, I strongly believe that these data should be provided to interpret the outliers in the results. Low age individuals have higher conc. of hair cortisol than adults. In addition corticosteroid-containing medication interfere in the measurements and the degree is depending on the method used.\n\nIs the rationale for developing the new method (or application) clearly explained? Partly\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [] }, { "id": "92503", "date": "06 Oct 2021", "name": "Gabriela Berg", "expertise": [ "Reviewer Expertise Clinical laboratory" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript by Reid et al., presents an option for reducing the number of steps in cortisol hair extraction by not transferring the sample from one to another container. It is always interesting to reduce possible errors and costs in this complex method. However, there are main limitations in the manuscript as presented:\nIntroduction: It is large. The first and second paragraph can be reduced to a shorter one. The reference to GR is not necessary in the manuscript context. The presentation of the aims in different paragraphs and with subtitles is not usual, and unless it is a requirement of the journal, it seems more like a project than a manuscript. Besides, the aims are not clear, what do the authors want to validate? Is it the cortisol extraction? It must be re-written according to the main objective.\nMethods:\nRegarding the reproducibility assay, the authors used the shaved hair from temporal and occipital regions of the participant’s head. This is not the recommended sample for cortisol hair assay, and this is the first limitation. Even though cortisol can be found in the hair from different areas of the head, it is very recommended to use the hair from the posterior vertex, so if the authors want to verify reproducibility, they must guarantee to have the correct sample to use.\n\nThe ELISA used in this manuscript is validated by the manufacturer for saliva cortisol measurement. It is a mistake to use this method for hair cortisol, unless the authors do the specific validation for using it on hair. As they use it in a different matrix than the one designed by the manufacturer, they should do a complete validation according to what is established by CLSI.\n\nIn the statistical analysis, the authors write “We opted instead to use non-parametric analysis…” - do the data present a parametric or non-parametric distribution? According to the distribution, determines the test to use. If the method shows extreme outliers in the replicates that increase the CV%, it must be taken into account. The weakness may be the use of a method not designed for the hair matrix, and in high values, it may not be precise.\nResults: Again, the presentation of the data must reflect their distribution. In Table 1, the authors choose to use mean and median to show the behavior of the data in Study 1, this is not correct but could be justified, at least, to show the behavior of the replicates; but in Study 2, it should be presented according to the correct statistical distribution of the data.\nDiscussion: The discussion is long and, in some paragraphs, confusing. The authors affirm that the method presents reproducibility, but they should apply the CLSI guidelines (EP-15A2) to confirm this. Besides, the use of the ELISA for saliva without validating for a different matrix shows limitations, the validation should be performed.\n\nIs the rationale for developing the new method (or application) clearly explained? Partly\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Partly\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? No", "responses": [] } ]	1	https://f1000research.com/articles/10-349
https://f1000research.com/articles/10-345/v1	05 May 21	{ "type": "Systematic Review", "title": "Demystifying media sources of information and levels of knowledge about COVID-19: a rapid mini-review of cross-sectional studies in Africa", "authors": [ "Dickson Aruhomukama", "Douglas Bulafu", "Douglas Bulafu" ], "abstract": "COVID-19 control is highly affected by knowledge levels which are also affected by receipt of information on the disease. Therefore, this review examined knowledge levels and media sources of information findings of the studies on knowledge, attitudes, perceptions, and practices towards COVID-19 done in low- and middle-income countries in Africa to shed light on the interplay of the use of different media platforms and populations’ knowledge about the COVID-19 pandemic and identify shorter- and longer-term priorities for COVID-19 research to improve the continent’s capacity to not only deal with COVID-19 but also future pandemics. Searches were conducted in PubMed and CINAHL databases/sites with major terms being “knowledge”, “attitudes”, “perceptions”, “practices”, “COVID-19 “, and “Africa”. 319 were where identified and subjected to the exclusion and inclusion criteria retaining only 10 free, full-text research articles related to knowledge, attitudes, perceptions, and practices towards COVID-19. This review summarized the levels of knowledge and media information sources among African populations. The review indicated a largely higher level of knowledge towards COVID-19 among populations who received information through various media platforms and alluded to the different media platforms that could be appropriate to spread COVID-19 related information to African populations.", "keywords": [ "COVID-19", "Knowledge levels", "Media information sources", "Africa" ], "content": "Introduction\n\nThe 2019 novel coronavirus disease (COVID-19) is a communicable respiratory disease caused by a novel strain of coronavirus, the Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2.1–4 COVID-19 was first identified in Wuhan, Hubei Province of China at the end of 2019 and was later declared a pandemic on the 11th of March 2020, underscoring the disease's high contagion potential, and exponential global rise.1–4\n\nAt the start of the pandemic, scholars had predicted that over 16.3 million people in Africa could have contracted COVID-19 by 30th June 2020.5 These predictions had been attributed to Africa’s rather weak health service infrastructure and low clinician to population ratio,6 limited laboratory capacity,7 and a higher rate of underlying conditions including malnutrition, anemia, HIV/AIDs, and chronic respiratory conditions due to tuberculosis and air pollution.8\n\nDespite Africa’s vulnerabilities, at the time of writing (i.e. 14:30 East African Time (EAT) on the 11th of November 2020), only 45/54 African countries had been hit by COVID-19 with only 1.3 million cumulative cases and 24,464 deaths.9 This could be attributed to the high levels of knowledge,10–12 positive attitudes and perceptions,11,12 and good practices towards COVID-1910,13–15 among the African people, as good knowledge, attitudes, perceptions, and practices have been reported to positively influence disease prevention as well as health promotion.16,17\n\nMany African countries put up stringent measures such as: instigating institutional and self-quarantines, restricted access to borders, closure of learning institutions, banning public transport and gatherings, among others to curb COVID-19.13 It is worth noting that for such measures to be effective, there should be public adherence which is affected by people’s levels of knowledge, which in turn influences their attitudes, perceptions, and practices.18,19\n\nScholars have also revealed that knowledge towards infectious diseases could be associated with serious panic alongside emotional reactions among people, which could hinder measures to curb the COVID-19 pandemic.20,21 These involve a range of opinions about the causes of the disease and exacerbating factors, identification of symptoms, and available methods of treatments and consequences that could determine different behaviors and preventive measures.22,23 Therefore, low levels of knowledge, towards COVID-19 and its preventive measures could be potential barriers in controlling the pandemic.17,22\n\nAlthough various studies on knowledge towards COVID-19 have been completed,13,24,25 no review has been pursued to interrogate and integrate the findings of these studies with regards to the levels of knowledge about COVID-19 and media sources of information about the same in low and middle-income countries in Africa.\n\nPursuing such a review would allow the: (i) deeper understanding of how the use of different media platforms affects knowledge which could constitute an important component for the implementation of COVID-19 prevention and control measures at both national and sub-national levels, (ii) identification of shorter- and longer-term priorities for COVID-19 research on the continent, and (iii) development of an agenda for both shorter- and longer-term priorities to help build Africa’s capacity to deal with future pandemics.\n\nHence, the objective of this review was to interrogate and integrate knowledge levels and media sources of information findings of the studies on knowledge, attitudes, perceptions, and practices towards COVID-19 done in low and middle-income countries in Africa to shed light on the interplay of the use of different media platforms and populations’ knowledge about the COVID-19 pandemic and identify shorter- and longer-term priorities for COVID-19 research which when pursued could improve the continent’s capacity to not only deal with COVID-19 but also future pandemics.\n\n\nMethods\n\nStudies that were included fitted the criteria below:\n\n1. Population; African population\n\n2. Intervention/Exposure of interest; Corona Virus Disease 2019\n\n3. Comparison; N/A\n\n4. Outcome; Knowledge, attitudes, practices, perception\n\n5. Study design; Cross Sectional studies\n\n6. Study period; January 2019 to October 2020\n\n7. Language; English\n\nFurthermore, studies that were not available in English, all other studies other than cross-sectional studies, studies not related to knowledge on COVID-19 will be excluded from the study. A protocol was not registered for this study.\n\nA literature search was conducted in from January 2019 to October 2020 (time of last search) on PubMed and CINAHL databases/sites with major terms being “knowledge”, “attitudes”, “perceptions”, “practices”, “COVID-19 “, and “Africa”. The search yielded a total of 305 articles at initial search in PubMed and 14 articles in CINAHL.\n\nSearch string: (((practices) OR (attitudes) OR (knowledge) OR (perceptions)) AND (Africa)) AND COVID-19.\n\nThe articles were moved to endnote where reviewers, DB and DA, screened articles basing on titles and abstracts. During this process; duplicates, systematic reviews, opinions, letters to editors, perspectives, and articles related to COVID-19 but not related to knowledge, attitudes, perceptions, and practices towards COVID-19 were removed, retaining only free, full-text research articles related to knowledge, attitudes, perceptions, and practices towards COVID-19 conducted among African countries, which were then considered for this review (Figure 1).\n\nAn extraction sheet was created in Microsoft word. One independent reviewer (DB) extracted the data and entries were checked by the second reviewer (DA). This extraction form included; study number, authors, study designs, country, respondents, number of respondents, and data collection tools.\n\nIn this review, two researchers (DB and DA) independently assessed the potential bias of the studies. Also, the Joana Briggs Institute Prevalence Critical Appraisal Tool, which is used for systematic reviews of prevalence studies, was used to assess the risk of bias in the studies [1]. Sample representativeness, sampling method, sample size, study participants, sites and settings, coverage of the identified sample, and appropriateness of the statistical analysis were evaluated in all included studies. Grading of Recommendations Assessment and Development Evaluation (GRADE) was used to assess the quality of evidence of the studies. Methodological quality, directness of the evidence, heterogeneity of data, and risk of publication bias were considered in the criteria. The outcomes were graded as low, moderate and high.\n\n\nResults\n\nThe initial literature search resulted into 319 articles (305 articles from PubMed and 14 articles from CINAHL). During the screening process, 309 articles did not meet the inclusion criteria and were excluded leaving 10 cross-sectional studies as shown in Figure 1.\n\n10 studies related to knowledge, attitudes, perceptions, and practices towards COVID-19 in African countries i.e., Egypt, Cameroon, Uganda, Nigeria, Libya, the Democratic Republic of Congo, Ethiopia, and Sierra Leone were reviewed. Only four of the studies used self-administered questionnaires while six studies used online forms to collect data during the pandemic. The respondents/participants in these studies included: the public/communities, medical students and lecturers, and health-care workers. The total number of respondents/participants involved in the studies were 8,013 (Table 1).\n\nThe Joana Briggs Institute Prevalence Critical Appraisal Tool, which is used for systematic reviews of prevalence studies, was used to assess the risk of bias in the studies. All the studies had a low risk of bias in terms of Sample representativeness, Methodological quality, coverage of the identified sample, and appropriateness of the statistical analysis, and risk of publication bias. Results of the GRADE analysis are available on Figshare.68\n\n10 studies featured results on knowledge with regards to COVID-19 among the: public/communities, medical students and lecturers, and health-care workers (Table 2).\n\nFour studies reported that nearly all of the respondents/participants had heard about COVID-19.13,15,26,28 Furthermore, three studies,10–12 reported that respondents/participants had adequate knowledge of COVID-19 while one study,27 reported that respondents/participants had inadequate knowledge of COVID-19.\n\nRespondents/participants in four studies,10,13,15,26 reported that social media platforms, for example, WhatsApp, Twitter and Facebook, local television and radio stations, and other internet platforms largely organizational websites were their major sources of information with regards to COVID-19. Respondents/participants in two studies,13,15 also reported having received information with regards to COVID-19 from their family members, friends as well as places of worship, for example mosques and churches (Table 2).\n\n\n\n• All, 100% of the participants reported having heard about COVID-19. The commonest reported sources of information about the disease were social media, 66.9%, the internet, 58.3%, and TV or satellite channels, 52.6%.\n\n\n\n• The majority, 84.19% of the respondents were reported to have had high knowledge scores of 4–7 regarding the modes of transmission of COVID-19. More than half, 54.5% reported having obtained information about the disease for the first time via television during the first and last 15 days of the study. The respondents also reported having obtained information about the disease through WhatsApp, 15.6%, and websites, 16.1%.\n\n\n\n• Nearly all, 99.7% of the participants reported having heard about COVID-19. The majority of the participants, 80.6% reported having heard and/or seen the messages on local television stations. Other sources of information about COVID-19 reported by the participants included: local radio stations, 64.3%, family and friends, 14.7%, local newspapers, 15.6%, social media platforms, 29.5%, and other internet platforms, 5.8%.\n\n\n\n• Most lecturers, 96%, and students, 92.5% reported that they knew about COVID-19 and its modes of transmission.\n\n\n\n• The majority, 73.5% of the participants reported that they had inadequate knowledge about COVID-19.\n\n\n\n• Most of the respondents, 61.6% reported that they had satisfactory knowledge about COVID-19. The respondents also reported that the internet as their main source of information about the disease.\n\n\n\n• A high proportion, 95.1% of the visitors reported that they knew about COVID-19 and its modes of transmission.\n\n\n\n• The majority of the participants, 97.7% in Ethiopia, and 99.5% in DRC reported that they knew about COVID-19 and its modes of transmission. The percentages of respondents with knowledge of COVID-19 modes of transmission by droplets were 98.8% in Ethiopia, and 94.9% in DRC. While about 48.1% of respondents in Ethiopia and 63.4% in DRC knew about airborne transmission. Those with knowledge of COVID-19 transmission via contaminated objects were 98.3% in Ethiopia, 93.4% in DRC.\n\n\n\n• The mean knowledge score of the participants was 13.1 (SD 1.2) indicating a good overall knowledge among medical students. The majority of medical students identified fever, cough, and difficulty in breathing as the main clinical symptoms of COVID-19 (95%, 85%, and 88%, respectively).\n\n\n\n• The majority, 91% of the participants reported that they had heard about COVID-19. The commonest reported sources of information about the disease were social media, 39%, radio, 73%, and churches/mosques, 24%. The majority of the participants mentioned body fluids, 74%, air, 61%, and touching, 66% infected persons or surfaces as the commonest modes of transmission of the disease.\n\n\nDiscussion\n\nTo the best of our knowledge, this is the first review of the levels of knowledge and media information sources about COVID-19 in Africa. This review identified shorter- and longer-term priorities for COVID-19 research on the continent, which when pursued could potentially improve the continent’s capacity to not only efficiently and effectively deal with COVID-19 but also future pandemics.\n\nSimilar to related studies, the high levels of knowledge about COVID-19 in some of the reviewed studies could be attributed to the seriousness of the disease in addition to the daily updates from public health agencies in the respective countries which could have positively influenced the respondents/participants need to learn and acquire knowledge about the disease,11,29 and the role of the different media platforms especially social media in explaining the basics about the disease.29–33\n\nWhile high knowledge levels have been documented to positively influence populations’ adherence to infection control and prevention measures,25,29 indifferent adherence to untrue and gullible beliefs by populations have been documented to habitually arise due to inadequate knowledge and inaccurate information, which could further affect the readiness levels as well as proper implementation of infection prevention and control measures at both the national or sub-national levels.25,29 Although social media platforms offer opportunities for specialists to speedily convey accurate information, they also offer other non-specialists opportunities to counter this with the spread of misinformation and exacerbating outrage.34,35 In concert, hazard and outrage along with socio-cultural and economic context shape adherence to, as well as the overall acceptance of, infection prevention and control measures.34–36\n\nStudies similar to those reviewed have reported the common use of social media platforms largely by the young adults and more educated attributing this to their understanding of the English language,29,37,38 while others have shown that social media platforms are also largely used by those having upper and middle socio-economic status,29,39 and urban and sub-urban dwellers40 who own smartphones and can easily access the internet or afford mobile data and as a result, the reviewed studies could have excluded the older adults, uneducated, those having low social-economic status, and rural dwellers.29 Unlike a study on smartphone internet access and use in the United States41 that reported the dependency on smartphones by similar groups of people (i.e. the uneducated and those having low social-economic status), despite the limited data about smartphone dependency in low- and middle-income countries in Africa,37 available indicators depict non-dependence.38 The same study41 however, showed that the individuals with high and middle-income status use smartphones and the internet as sources of news and/or information. Indeed, the findings of the reviewed studies indicate that smartphones could potentially be acting as barriers for disadvantaged groups to overcome the digital divide.\n\nAlso, unlike the findings of another study on political attitudes and demographics of British social media users that reported no statistically significant differences between social media users and non-social media users after controlling for age, gender, and education,42 similar studies on the utilization of social media for health-related purposes in low- and middle-income countries in Africa have reported otherwise.11,29,37,38,43 Above and beyond, the utilization of social media platforms for health-related purposes in low- and middle-income countries in Africa has been reported to be on the increase.37,38,44\n\nWith the projected rise in smartphone ownership and internet usage in low- and middle-income countries in Africa,44 the findings of the reviewed studies implore the use of social media platforms as tools to wisely, prudently, and speedily spread the constantly changing information about COVID-19. Social media platforms when used as tools could serve to change people’s behavior as well as to promote the well-being of individuals and public health. Despite this, the role of social media in shaping knowledge, attitudes, perceptions, and practices should be carefully studied to increase compliance with infection prevention and control measures at both the national or sub-national levels. Also, the understanding of varying opinions and concerns of different demographic groups could be done to enable public health officials to design and implement on-target response strategies. In a similar light, research is needed to expand the geographical focus and test strategies to facilitate the efficient and effective use of social media for health-related purposes in low- and middle-income countries in Africa.\n\nAlso, the role of public health officials should be applauded and fortified, while a framework to improve the public health emergency preparedness system is pursued to encourage a focused conversation to improve preparedness for the benefit of individuals, families, communities, and societies.\n\nThe low levels of knowledge about COVID-19 reported in the reviewed studies could be attributed to several factors reported in related studies including misinformation powered by rumors, stigma, and several conspiracy beliefs about the disease,45,46 low levels of education in most of the populations in the African countries in which the studies were pursued,47–49 lack of access to information as well as health services,50–52 low perceived risk of acquiring the disease,53,54 and other gaps in socio-demographic groupings among the populations, for example age and socio-economic groupings.11,29,37,38,43\n\nMisinformation related to COVID-19 has been reported to be gaining considerable popularity and playing a role in augmenting the disease’s threat through encouraging its continuous global spread.46,55,56 Also, misinformation can negatively impact individuals’, communities’, and societies’ actions, and devalue infection prevention and control measures employed at both the national or sub-national levels.46 Misinformation has also been documented to potentially have serious negative implications not only on individuals and communities but also societies particularly if prioritized over scientifically proven guidelines.46,55,56 Maintaining effective information governance across the general public to substitute any misinformation related to the disease especially in Africa has been recommended even though falsehoods on various media platforms have been predicted to continue surging high as long as the disease lingers on.46 The findings of the reviewed studies sustain the suggestions that regular prompts on the concept of accuracy by several mass media platforms be given as these may not only enhance individuals’, communities’ and societies’ sharing decisions related to COVID-19 but could also ease the volume of misinformation about the disease.46,57 The findings of the reviewed studies also suggest the need for health agencies to trail misinformation related to COVID-19 possibly in real-time, and to involve individuals, communities, and societies at large to demystify misinformation.\n\nRegarding the gaps in socio-demographic groups, the use of more native non-smartphone and/or non-internet requiring mass media campaigns through which high proportions of large populations can be exposed to COVID-19 related messages via television, radio, and non-digital or traditional newspaper platforms could be explored to target especially individuals of low- socioeconomic status, the elderly as well as those dwelling in rural settings. This assertion is based on previous studies on how rural communities accessed and used information that had been conducted in rural communities, these studies revealed that most of the information needed by these populations related to day-to-day problems, and that most of the information was accessed mainly through radio, television, and traditional or non-digital newspapers, as well as face to face communication.58–60 These studies further revealed that the choice of information sources was majorly influenced by among other factors the levels of education, income, as well as the occupation of their respondents.58–60\n\nThe same assertion is supported by the findings of related studies that investigated sources of COVID-19 related information that revealed that populations constituting of mainly the older adults, unemployed or those mainly employed in the informal sector, uneducated, and rural and semi-urban dwellers have limited or poor knowledge which could be associated with negative attitudes, perceptions, and practices towards COVID-19.29,61 The findings of the reviewed studies could enhance the recommendations previously made by several related studies,29,61 that knowledge, attitude, and practices towards COVID-19 studies be pursued especially in the underprivileged and vulnerable groups especially as the COVID-19 pandemic lingers on.\n\nAlthough exposure to messages through mass media campaigns has been described as largely passive, with such campaigns recurrently competing with several factors such as prevailing social norms, persistent merchandise marketing, and behaviors driven by habit and/or addiction.62 Through the evaluation of the same campaigns in the context of various health risk behaviors, mass media campaigns have been documented to have the potential to positively influence health-related behavior, particularly across large populations.62 The findings of this study in the context of the reviewed studies hence suggest that mass media campaigns could be leveraged to spread COVID-19 related information and that investments in longer and better-funded mass media campaigns to achieve adequate population exposure to media messages should be done.\n\nWith regards to face-to-face communication, several avenues have been identified through studies related to those reviewed including the utilization of small and regulated meetings of different religious faiths and music concerts, engaging community health workers in spreading COVID-19 related messages in their communities as well as encouraging interpersonal communication in families aimed at spreading information about COVID-19.63–66 Communication of health-related themes through music has been recognized to foster a culture-centered approach that considers community participation concerning broader social and organizational issues as compared to individual-focused behavior change communication. Also, communication of health-related themes through music has been reported to have the potential to bridge and build trust between health care workers particularly community health care workers, and target communities while facilitating information dissemination as well as stimulating public debate regarding sensitive health-related themes.40\n\n\nConclusion\n\nThis review largely indicates higher levels of knowledge about COVID-19 among populations in low- and middle-income settings in Africa that had received information about the same via several media platforms. In populations with lower levels of knowledge about COVID-19, this review alludes to the different media platforms that could be appropriate to spread COVID-19 related information to the same populations. The ability of health care agencies to communicate COVID-19 related messages while leveraging appropriate media platforms can play a critical role in easing the shorter- and longer-term impacts of the COVID-19 pandemic in low- and middle-income settings in Africa.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nFigshare: GRADE results_Demystifying media sources of information and levels of knowledge about COVID-19-a rapid mini-review of cross-sectional studies in Africa. https://doi.org/10.6084/m9.figshare.14160758.v1.68\n\nFigshare: PRISMA checklist for ‘Demystifying media sources of information and levels of knowledge about COVID-19: a rapid mini-review of cross-sectional studies in Africa.’ https://doi.org/10.6084/m9.figshare.14045471.v1.67\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nCompeting interests\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe authors declared that no grants were involved in supporting this work.", "appendix": "References\n\nOrnell F, Schuch JB, Sordi AO, et al.: “Pandemic fear” and COVID-19: mental health burden and strategies. 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Publisher Full Text\n\nKarasneh R, et al.: 2020; Media’s effect on shaping knowledge, awareness risk perceptions and communication practices of pandemic COVID-19 among pharmacists. Res Social Adm Pharm. 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIslam AKMN, Laato S, Talukder S, et al.: Misinformation sharing and social media fatigue during COVID-19: An affordance and cognitive load perspective. Technol. Forecast. Soc. Change. 2020; 159: 120201. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMalecki K, Keating JA, Safdar N: Crisis communication and public perception of COVID-19 risk in the era of social media. Clin. Infect. Dis. 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUmviligihozo G, et al.: Sub-Saharan Africa preparedness and response to the COVID-19 pandemic: A perspective of early career African scientists. Wellcome open Res. 2020; 5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHagg E, Dahinten VS, Currie LM: The emerging use of social media for health-related purposes in low and middle-income countries: A scoping review. Int. J. Med. Inform. 2018; 115: 92–105. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSilver L, Johnson C: Internet use is growing across much of sub-Saharan Africa, but most are still offline. Pew Res. 2018Reference Source\n\nPreoţiuc-Pietro D, Volkova S, Lampos V, et al.: Studying user income through language, behaviour and affect in social media. PLoS One. 2015; 10: e0138717. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHuang Q, Wong DWS: Activity patterns, socioeconomic status and urban spatial structure: what can social media data tell us? Int. J. Geogr. Inf. Sci. 2016; 30: 1873–1898. Publisher Full Text\n\nTsetsi E, Rains SA: Smartphone Internet access and use: Extending the digital divide and usage gap. Mob. Media Commun. 2017; 5: 239–255. Publisher Full Text\n\nMellon J, Prosser C: Twitter and Facebook are not representative of the general population: Political attitudes and demographics of British social media users. Res. Polit. 2017; 4: 2053168017720008. Publisher Full Text\n\nShava H, Chinyamurindi WT: Determinants of social media usage among a sample of rural South African youth. South African J. Inf. Manag. 2018; 20: 1–8. Publisher Full Text\n\nPoushter J: Smartphone ownership and internet usage continues to climb in emerging economies. Pew Res. Cent. 2016; 22: 1–44.\n\nOlatunji OS, Ayandele O, Ashirudeen D, et al.: “Infodemic” in a pandemic: COVID-19 conspiracy theories in an african country. Soc. Heal. Behav. 2020; 3: 152. Publisher Full Text\n\nAhinkorah BO, Ameyaw EK, Hagan JE Jr, et al.: Rising above misinformation or fake news in Africa: Another strategy to control COVID-19 spread. Front. Commun. 2020; 5: 45. Publisher Full Text\n\nRakhmanov O, Dane S: Knowledge and anxiety levels of African university students against COVID-19 during the pandemic outbreak by an online survey. J Res Med Dent. Sci. 2020; 8: 53–56.\n\nAdesegun OA, et al.: The COVID-19 crisis in Sub-Saharan Africa: Knowledge, attitudes, and practices of the Nigerian public. Am. J. Trop. Med. Hyg. 2020; 103: 1997–2004. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBates BR, Moncayo AL, Costales JA, et al.: Knowledge, attitudes, and practices towards COVID-19 among Ecuadorians during the outbreak: an online cross-sectional survey. J. Community Health. 2020; 45: 1158–1167. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShadmi E, et al.: Health equity and COVID-19: global perspectives. Int. J. Equity Health. 2020; 19: 1–16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAsemahagn MA: Factors determining the knowledge and prevention practice of healthcare workers towards COVID-19 in Amhara region, Ethiopia: a cross-sectional survey. Trop. Med. Health. 2020; 48: 1–11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAustrian K, et al.: COVID-19 related knowledge, attitudes, practices and needs of households in informal settlements in Nairobi, Kenya.2020.\n\nSubramaney U, et al.: Coronavirus disease 2019 (COVID-19) and psychiatric sequelae in South Africa: Anxiety and beyond. Wits J. Clin. Med. 2020; 2: 115–122. Publisher Full Text\n\nBanda J, et al.: Knowledge, risk perceptions and behaviors related to the COVID–19 pandemic in Malawi.Publisher Full Text\n\nVraga EK, Tully M, Bode L: Empowering users to respond to misinformation about Covid-19. Media Commun. 2020; 8: 475–479. Publisher Full Text\n\nMian A, Khan S: Coronavirus: the spread of misinformation. BMC Med. 2020; 18: 1–2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPennycook G, McPhetres J, Zhang Y, et al.: Fighting COVID-19 misinformation on social media: Experimental evidence for a scalable accuracy-nudge intervention. Psychol. Sci. 2020; 31: 770–780. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMtega WP: Access to and usage of information among rural communities: A case study of Kilosa District Morogoro Region in Tanzania. Partnersh. Can. J. Libr. Inf. Pract. Res. 2012; 7. Publisher Full Text\n\nHutchinson P, Lance P, Guilkey DK, et al.: Measuring the cost-effectiveness of a national health communication program in rural Bangladesh. J. Health Commun. 2006; 11: 91–121. PubMed Abstract \| Publisher Full Text\n\nVan Rossem R, Meekers D: The reach and impact of social marketing and reproductive health communication campaigns in Zambia. BMC Public Health. 2007; 7: 352. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nByanaku A, Ibrahim M: Knowledge, attitudes, and practices (KAP) towards COVID-19: A quick online cross-sectional survey among Tanzanian residents. medRxiv .2020. Publisher Full Text\n\nWakefield MA, Loken B, Hornik RC: Use of mass media campaigns to change health behaviour. Lancet. 2010; 376: 1261–1271. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAtaguba OA, Ataguba JE: Social determinants of health: the role of effective communication in the COVID-19 pandemic in developing countries. Glob. Health Action. 2020; 13: 1788263. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nModell SM, Kardia SLR: Religion as a health promoter during the 2019/2020 COVID outbreak: View from Detroit. J. Relig. Health. 2020; 59: 2243–2255. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNúñez A, Madison M, Schiavo R, et al.: Responding to healthcare disparities and challenges with access to care during COVID-19. Heal. Equity. 2020; 4: 117–128. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcConnell BB: Music and health communication in The Gambia: A social capital approach. Soc. Sci. Med. 2016; 169: 132–140. PubMed Abstract \| Publisher Full Text\n\nAruhomukama D: Demystifying media sources of information and levels of knowledge about COVID-19: a rapid mini-review of cross-sectional studies in Africa. figshare. 2021. Publisher Full Text\n\nAruhomukama D, Bulafu D: GRADE results_Demystifying media sources of information and levels of knowledge about COVID-19-a rapid mini-review of cross-sectional studies in Africa. figshare. 2021. Publisher Full Text\n\nMunn Z, Moola S, Riitano D, et al.: The development of a critical appraisal tool for use in systematic reviews addressing questions of prevalence. Int J Health Policy Manag. 2014; 3(3): 123. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "86712", "date": "04 Jun 2021", "name": "Zohra S. Lassi", "expertise": [ "Reviewer Expertise Systematic review methodology." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for giving me an opportunity to review this paper.\n\nThe search was last conducted in October 2020 which is too old and needs to be updated as much literature has been published since then.\n\nI think the Exposure/Intervention should be a Digital platform for COVID-19 information.\n\nHow are self-administered questionnaires different from online? I believe online questionnaires were also self-administered. Please clarify?\n\nAlthough the reviewers have discussed each platform in detail in the discussion section, I guess the importance of authentic source information and pros/cons of using each platform and their impact on population's mental health should be discussed.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Partly\n\nIs the statistical analysis and its interpretation appropriate? Partly\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [ { "c_id": "6763", "date": "09 Jul 2021", "name": "Dickson Aruhomukama", "role": "Author Response", "response": "We thank the reviewer for the great comments, We will respond to each of them in the revised version of the write-up." } ] }, { "id": "85999", "date": "04 Jun 2021", "name": "Ahmed Shihab Albahri", "expertise": [ "Reviewer Expertise Tele-medicine", "healthcare services", "bioinformatics", "artificial intelligence" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe corrections and comments are based on the manuscript.\nConcern number 1: I think the problem of this study is questionable and not valid. The authors reported: “COVID-19 control is highly affected by knowledge levels which are also affected by receipt of information on the disease.” However, in my opinion, this is not a hot topic problem to justify the presented systematic study. It could be a part of the contributions rather than the main problem. Another way to solve this issue is to present the advanced idea that matches receipt of the information on the COVID-19 disease problem.\n\nConcern number 2: It is advisable that the abstract contains a statement at the end with the main achievement/contribution of the paper at a higher level and how this might be useful overall.\n\nConcern number 3: According to the above concerns, reorganize the abstract to conclude: (a) The overall purpose of the paper and the research problems you investigated including the background and the contexts. (b) The basic design of the study. (c) Major findings or trends found as a result of the study. (d) A summary of your interpretations and conclusions.\n\nConcern number 4: I need the authors to explain why this study includes only 2 databases, in particular, the author mentioned “A literature search was conducted in from January 2019 to October 2020 (time of last search) on PubMed and CINAHL databases/sites”. In this context, more reliable databases should be included to collect more articles due to the limited number of studies (only 10 studies).\n\nConcern number 5: In general, the discussion is confusing to the readers. The discussion presented paragraph after paragraph and neither summary points nor claim points were explaining the topic of the paper. Although some interesting discussed subjects were presented the overall direction of this section is confusing and not apparent.\n\nConcern number 6: The conclusion is also confusing in general. So rewrite it and consider the following comments: -\n\nHighlight your analysis and reflect only the important points for the whole paper. -\n\nMention the benefits. -\n\nMention the implication in the last part of this section.\n\nConcern number 7: In conclusion, my question here is: how can the readers be satisfied with the presented study without a clear justification from the academic literature about challenges, limitations, gaps…etc. Therefore, and based on the above, I reject the paper for indexing. However, some comments that could improve the paper are presented above.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Partly\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required.\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-345
https://f1000research.com/articles/9-1437/v1	10 Dec 20	{ "type": "Study Protocol", "title": "Determinants of in-hospital death in patients with a thrombus straddling a patent foramen ovale: protocol of a systematic review", "authors": [ "Amado Jiménez-Ruiz", "Palak Shah", "Andrew Gibson", "Juan Camilo Vargas-González", "Luciano A. Sposato", "Palak Shah", "Andrew Gibson", "Juan Camilo Vargas-González", "Luciano A. Sposato" ], "abstract": "Background: Thrombi identified on echocardiography at the time of straddling a patent foramen ovale (PFO) constitute a medical emergency with an associated imminent risk of death. Ischemic stroke (IS) and myocardial infarction (MI) occurring in patients with a thrombus straddling a PFO (TSPFO) may be associated with increased risk of in-hospital death. Variables associated with increased risk of death in women and men may be different. We will perform a systematic review of case reports and cases series of patients with a TSPFO to assess if IS and MI are associated with increased risk of in-hospital death and we will further stratify analyses by sex. Methods: This systematic review will include all case reports and case series of adult patients (18-year-old or older) with echocardiographic or pathological (e.g. at autopsy for older reports) evidence of a TSPFO published between inception and June 30, 2020, in any language. We will search in PubMed and Embase databases. Two reviewers will independently screen titles and abstracts, retrieve full texts, and extract the data in a predesigned form. We will apply a multivariable logistic regression analysis to estimate the association of IS and MI with in-hospital mortality. We will stratify analyses by sex. Discussion: IS and MI in patients with TSPFO could potentially be associated with worse outcomes if they are not timely identified or left untreated. Both acute IS and MI require specific treatment (e.g. thrombolysis, primary coronary intervention, or mechanical thrombectomy) that may be influenced by the therapy instituted for the TSPFO. Knowing the incidence of acute IS and MI among patients diagnosed with TSPFO and whether they are associated with an increased risk of death would help to improve the management of this medical emergency. Protocol registration: CRD42020216118, PROSPERO.", "keywords": [ "Stroke", "myocardial infarction", "patent foramen ovale", "paradoxical embolism", "death", "mortality", "thrombus in transit" ], "content": "Introduction\n\nA patent foramen ovale (PFO) is a common finding, present in 25% of individuals on autopsy1 and frequently reported as an incidental finding on echocardiography studies2. Most patients are asymptomatic, and the presence of a PFO is usually considered clinically irrelevant. However, a thrombus can sometimes be found straddling a PFO in the context of acute pulmonary embolism, deep venous thrombosis, acute respiratory insufficiency, acute coronary syndromes, or acute ischemic stroke (IS)3.\n\nA thrombus straddling a PFO (TSPFO) carries a high risk of impending paradoxical embolism and death4. Most of the evidence on the diagnosis and treatment of TSPFO comes from anecdotal single case reports or small case series4. As such, factors associated with increased risk of death are unknown. Similarly, there is clear guidance on what the best treatment approach is for TSPFO. Paradoxical embolism is a relatively common cause of IS in patients with PFO and could possibly explain the high mortality in patients with a TSPFO5. Although less frequently reported, paradoxical embolism has also been identified as a cause of acute myocardial infarction (MI). Both IS and MI are well recognized causes of death among the general population. We hypothesized that IS and MI identified at the time of the diagnosis of a TSPFO are associated with increased risk of in-hospital death. We will therefore systematically review all case reports and case series of patients with TSPFO published in the medical literature to assess whether IS, MI, and different therapeutics (e.g. anticoagulation, thrombolysis or surgical removal of the TSPFO) are associated with the adjusted risk of in-hospital death. We will also stratify these analyses by sex.\n\n\nMethods/design\n\nThe primary objective is to assess whether IS and MI at presentation are independently associated with the adjusted risk of in-hospital death in patients with a TSPFO. The secondary objective is to assess whether the association between IS and MI at presentation and the risk of in-hospital death in patients with a TSPFO varies in women and men.\n\nThis study protocol has been prepared according to the 2015 Preferred Reporting Items for Systematic Reviews, and Meta-Analyses Protocols (PRISMA-P) guidelines. We will use the PRISMA flowchart. We have submitted this systematic review to the International Prospective Register for Systematic Reviews and Meta-analysis (PROSPERO, registration number: CRD42020216118).\n\nWe will search PubMed and Embase databases to identify potentially eligible studies by applying predefined search terms (Table 1 and Table 2), published from inception to June 30, 2020 in any language.\n\nThree reviewers will independently screen titles and abstracts and will solve disagreements by consensus (AJR, PS, AG). The same reviewers will thoroughly assess all potentially relevant full texts and will document the reasons for excluding specific publications. We will include studies fulfilling all inclusion criteria and no exclusion criteria. Inclusion criteria will be: (a) case reports or case series; (b) adult patients (≥18 years-old); (c) complete data on in-hospital outcomes (e.g. dead or alive); and (d) complete demographic data (e.g. age and sex) and information on comorbidities, risk factors and acute treatment. Exclusion criteria include: (a) editorial or review articles; (b) duplicate reports; and (c) publications in which data at the patient-level is unavailable.\n\nThree independent reviewers will be used for selecting studies through each phase of the review (screening, eligibility, and inclusion meta-analysis). We will create and use a standardized Microsoft Forms data extraction form and Excel spreadsheet, extracting the following data from published reports: study identification (year of publication, first author); study characteristics (number of cases, continent where the study was conducted); patients' characteristics (age, sex), risk factors and comorbidities (obesity, hypertension, diabetes mellitus, hyperlipidemia, known atrial fibrillation, coronary artery disease, obstructive sleep apnea, cancer, chronic kidney disease, deep vein thrombosis, pulmonary embolism, transient ischemic attack, stroke, autoimmune disease); acute diagnoses upon admission (acute MI, acute IS, transient ischemic attack, hemorrhagic stroke, pulmonary embolism, deep vein thrombosis, syncope, peripheral paradoxical embolism, newly diagnosed atrial fibrillation); presenting symptoms (dyspnea, chest pain, palpitations, dizziness, syncope, focal neurological deficit, shock, loss of consciousness or coma, seizures, peripheral embolism); laboratory parameters (D-Dimer value, fibrinogen, brain natriuretic peptide, cardiac troponin); main affected coronary artery in patients with MI; vascular territory involved in patients with cerebrovascular events; organ involved in patients with peripheral embolism; most likely cause of venous thromboembolism (unprovoked, thrombophilia, cancer, trauma, post-operative, immobilization, pregnancy, recent flight, infection, other); cardiac and pulmonary investigations (transthoracic echocardiogram, transesophageal echocardiogram, computed angiography of the lungs, cardiac magnetic resonance imaging, cardiac computed tomography, electrocardiogram); main electrocardiographic findings (S1Q3T3 pattern, sinus tachycardia, other); echocardiographic findings [left ventricular dysfunction, right ventricular dysfunction, right ventricular function not reported, increased pulmonary artery pressure (>20 mmHg), dilated right ventricle, dilated right atrium]; acute treatment (intravenous thrombolysis, surgery, anticoagulation); in-hospital outcomes (full recovery, partial recovery, death); secondary prevention treatment (Aspirin or other antiplatelet agent, vitamin K antagonists, non-specified oral anticoagulants, direct oral anticoagulants, low molecular weight heparin, unfractionated heparin, PFO closure, Inferior vena cava filter); and secondary prevention outcome (no events, recurrent venous thromboembolism, incident/recurrent stroke or transient ischemic attack, recurrent/incident peripheral embolism, recurrent/incident MI, death).\n\nWe will conduct univariate analyses comparing the patients who died or survived during hospital stay. Variables with a p-value of <0.05 and those known to influence death in patients with venous thromboembolism, regardless of their level of significance on univariate in the analysis, will be included in a multivariable logistic regression for in-hospital death. We will use a random intercept model to account for the potential clustering effect of the different decades on outcomes. We will initially include all the potential covariables and we will subsequently perform a backward stepwise selection until fulfilling a prespecified level of significance of 0.05 for potential covariables. We will keep sex and age in the model because of being recognized confounders for death. We will conduct all analyses with R version 3.6.2.\n\nWe will apply the tool originally proposed by Murad et al. for assessing the methodological quality and synthesis of case series and case reports6 (domains, selection, ascertainment, causality, and reporting).\n\nWe do not anticipate any amendment to this review protocol. If an amendment is needed, we will document it and report it in a timely manner.\n\n\nEthics and dissemination\n\nThis systematic review will be based on published data. As such, it is not subject to ethical approval. The results will be published in peer-reviewed journals and presented at scientific conferences. All data underlying the results will be made available upon reasonable request.\n\n\nDiscussion\n\nIn the context of more available point of care echocardiography, TSPFO are expected to be increasingly reported among patients seen in the Emergency Department and intensive care unit for acute onset respiratory failure, shock or acute coronary syndromes7,8. The timely diagnosis of a TSPFO could radically influence acute treatment options and could provide critical information on potential patient outcomes. IS and MI can likely impact on the prognosis of patients with TSPFO, and are well-recognized complications of PFO in patients with pulmonary embolism and associated with increased risk of death9. Paradoxical embolism to the coronary arteries has been reported less frequently and its incidence remains undetermined3. Data on potential therapeutic interventions for patients with TSPFO are also scarce. A previous systematic review including 174 patients found a 35% lower 30-day mortality among surgically treated patients, although this was non-significant4. Knowing the incidence of IS and MI in patients with TSPFO and their association with in-hospital death, as well as outcomes of different therapeutic interventions would be important for improving awareness about prognostic factors and treatment options in this population.\n\n\nStudy status\n\nPreliminary searches have been carried out in PubMed and Embase databases.\n\n\nRegistration\n\nThis review protocol has been submitted to the International Prospective Register for Systematic Reviews and Meta-analysis (PROSPERO, registration number: CRD42020216118).\n\n\nData availability\n\nNo data is associated with this article.\n\nFigshare: PRISMA-P checklist for ‘Determinants of in-hospital death in patients with a thrombus straddling a patent foramen ovale: protocol of a systematic review’, https://doi.org/10.6084/m9.figshare.13281242.v110.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nHagen PT, Scholz DG, Edwards WD: Incidence and size of patent foramen ovale during the first 10 decades of life: an autopsy study of 965 normal hearts. Mayo Clin Proc. 1984; 59(1): 17–20. PubMed Abstract \| Publisher Full Text\n\nKoutroulou I, Tsivgoulis G, Tsalikakis D, et al.: Epidemiology of Patent Foramen Ovale in General Population and in Stroke Patients: A Narrative Review. Front Neurol. 2020; 11: 281. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAggarwal K, Jayam VK, Meyer MA, et al.: Thrombus-in-transit and paradoxical embolism. J Am Soc Echocardiogr. 2002; 15(9): 1021–1022. PubMed Abstract \| Publisher Full Text\n\nMyers PO, Bounameaux H, Panos A, et al.: Impending paradoxical embolism: systematic review of prognostic factors and treatment. Chest. 2010; 137(1): 164–170. PubMed Abstract \| Publisher Full Text\n\nJungbluth A, Erbel R, Darius H, et al.: Paradoxical coronary embolism: case report and review of the literature. Am Heart J. 1988; 116(3): 879–885. PubMed Abstract \| Publisher Full Text\n\nMurad MH, Sultan S, Haffar S, et al.: Methodological quality and synthesis of case series and case reports. BMJ Evid Based Med. 2018; 23(2): 60–63. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKuriakose D, O’Mahony R, Rooplalsingh R, et al.: Point of Care Echocardiography in an Irish Critical Care Unit. Ir Med J. 2018; 111(8): 800. PubMed Abstract\n\nChenkin J, Atzema CL: Contemporary Application of Point-of-Care Echocardiography in the Emergency Department. Can J Cardiol. 2018; 34(2): 109–116. PubMed Abstract \| Publisher Full Text\n\nLe Moigne E, Timsit S, Salem DB, et al.: Patent Foramen Ovale and Ischemic Stroke in Patients With Pulmonary Embolism: A Prospective Cohort Study. Ann Intern Med. 2019; 170(11): 756–763. PubMed Abstract \| Publisher Full Text\n\nShah P, Jiménez-Ruiz A, Gibson A, et al.: PRISMA-P-checklist. Determinants of in-hospital death in patients with a thrombus straddling a patent foramen ovale: protocol of a systematic review. figshare. Online resource. 2020. http://www.doi.org/10.6084/m9.figshare.13281242.v1" }	[ { "id": "80248", "date": "08 Mar 2021", "name": "Paolo Eusebi", "expertise": [ "Reviewer Expertise clinical trials", "meta-analysis", "diagnostics", "data visualization" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nWhy a meta-analysis of case reports and case series? Are other studies not available?\n\nPlease correct covariable with covariate.\n\nPlease consider adding penalized regressions like lasso (R package glmnet) in the methods. It could be a good option in the case of a small number of events per predictor.\n\nPlease, consider cross-validation techniques.\n\nInstead of targeting significance for the covariates, it would be better to target overall accuracy in predictions or AIC.\n\nIs the rationale for, and objectives of, the study clearly described? Partly\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "6446", "date": "16 Mar 2021", "name": "Amado Jiménez-Ruiz", "role": "Author Response", "response": "Dear Dr. Paolo Eusebi, First, we want to thank you for considering our manuscript and the time you and the reviewers devoted to reading it. We have received the feedback and suggestions, and we are addressing them point by point. We have modified the manuscript according to the comments below. 1. Why a meta-analysis of case reports and case series? Are other studies not available? Impending paradoxical embolism is an uncommon condition rarely reported in the medical literature. Our pilot exercise for the review concluded that the published information is limited to case reports and small case series and therefore based our data extraction plan and further analysis on these results. 2. Please correct covariable with covariate. We have corrected covariable to covariate as stated. 3. Please consider adding penalized regressions like lasso (R package glmnet) in the methods. It could be a good option in the case of a small number of events per predictor. During the process of analysis and selection of potential covariables for the project, we considered different methods. Some of those models were Ridge and LASSO. However, when we thought about our problem, we conclude that some variables should be in the model irrespective of their statistical significance (age and sex). In the case of LASSO and its autonomous nature for covariates for model selection, we could not ensure that the model would include both. Also, we performed multiple imputations through MICE; we cannot find a universal method to combine both techniques. For these two reasons, we selected a traditional stepwise approach. 4. Please, consider cross-validation techniques. We appreciate the suggestion to use cross-validation. However, to manage uncertainty, we decided to perform sensitivity analysis using a multilevel model and the scenario of cases with complete data. We decided this because our target audience (cardiologists and neurologists) is more familiar with sensitivity analysis. 5. Instead of targeting significance for the covariates, it would be better to target overall accuracy in predictions or AIC. We appreciate the suggestion of using the AIC to select the best model. It is explicitly stated in the protocol now. Thanks again for taking the time to review this manuscript." }, { "c_id": "6602", "date": "05 May 2021", "name": "Amado Jiménez-Ruiz", "role": "Author Response", "response": "Dear Dr. Paolo Eusebi, First, we want to thank you for considering our manuscript and the time you and the reviewers devoted to reading it. We have received the feedback and suggestions, and we are addressing them point by point. We have modified the manuscript according to the comments below. Why a meta-analysis of case reports and case series? Are other studies not available? Impending paradoxical embolism is an uncommon condition rarely reported in the medical literature. Our pilot exercise for the review concluded that the published information is limited to case reports and small case series and therefore based our data extraction plan and further analysis on these results. Please correct covariable with covariate. We have corrected covariable to covariate as stated. Please consider adding penalized regressions like lasso (R package glmnet) in the methods. It could be a good option in the case of a small number of events per predictor. During the process of analysis and selection of potential covariables for the project, we considered different methods. Some of those models were Ridge and LASSO. However, when we thought about our problem, we conclude that some variables should be in the model irrespective of their statistical significance (age and sex). In the case of LASSO and its autonomous nature for covariates for model selection, we could not ensure that the model would include both. Also, we performed multiple imputations through MICE; we cannot find a universal method to combine both techniques. For these two reasons, we selected a traditional stepwise approach. Please, consider cross-validation techniques. We appreciate the suggestion to use cross-validation. However, to manage uncertainty, we decided to perform sensitivity analysis using a multilevel model and the scenario of cases with complete data. We decided this because our target audience (cardiologists and neurologists) is more familiar with sensitivity analysis. Instead of targeting significance for the covariates, it would be better to target overall accuracy in predictions or AIC. We appreciate the suggestion of using the AIC to select the best model. It is explicitly stated in the protocol now. Thanks again for taking the time to review this manuscript. Sincerely, The Authors" } ] }, { "id": "80694", "date": "29 Mar 2021", "name": "Leonardo Varotto", "expertise": [ "Reviewer Expertise Clinical trials", "meta-analysis", "interventional cardiology", "interventional cardio-neurology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIs the rationale for, and objectives of, the study clearly described?\nYes, the importance of the study (the rationale that \"promotes\" it) is clear.\nIs the study design appropriate for the research question?\nPartially or, more properly, no!\nAre sufficient details of the methods provided to allow replication by others?\nYes, with the limitations mentioned below.\nAre the datasets clearly presented in a useable and accessible format?\nYes, but this aspect cannot be currently fully judged.\n\nThank you for the opportunity to review this manuscript. From my point of view, the study design should better develop the following points that I would like to resume briefly as below:\n1st problem: The patients who will constitute the matrix and the data do not come from randomized studies, but from publications-observational studies and for this reason \"pre-selected\" in order to \"promote a certain point of view\" concerning, for example, a therapy, etc… Patient selection (not the selection made by the authors) constitutes a heavy source of bias. Is it not possible to conduct the study by aggregating multiple therapy centers (hospitals) with which to agree on a therapeutic and screening protocol? (see next point)…\n\n2nd problem: The search for co-morbidities, and in general the patient's health state and risk factors, is truly “refined” and timely. However, the absence of one of the information to be acquired in a specific patient does not mean that it was absent. Unfortunately, given that the patients do not come from the same study cohort with an a priori standardized observation protocol, it does not guarantee that the information collected is homogeneous. In the meta-analyzes conducted by aggregating randomized studies, this problem is addressed with graphic tools (funnel plot), or with in-depth analyzes on the heterogeneity of the studies. In summary, in this research plan (study design) not a word is spent on how to overcome these serious and typical problems of heterogeneity of patients connected to the purposes of the publication and to the different way in which they have been screened at the moment, for example, of hospitalization.\n\n3rd problem: In the data analysis techniques (logistic model) the search for interaction between the factors that could affect the probability of death in hospital of the patient does not appear and, moreover, using the \"backward stepwise selection\" strategy the first part of the univariate analysis becomes superfluous. It is not wrong to use a logistic regression model (although for such models it is always difficult to offer an overall picture of the quality of the obtained result) but I would also use an analysis technique such as the \"random forest\" which could probabilize patients with particular co-morbidities or risk factors. Of the logistic model, it is necessary to present also quality indicators: wrong classification, pseudo R2, Nagelkerke indicator. These are fundamental indicators to give credibility to the results obtained.\n\n4th problem: It is not clear how many patients could be analyzed. The author described a previous systematic review with 174 patients analysed, a number apparently very limited to contain measurement errors and to offer adequate power to statistical tests. What is the information gathering forecast?\n\nI hope that the authors are encouraged by my remarks made with a constructive aim because it is very important to carry on such typology of studies. For this reason, it would be desirable that authors will accomplish their hard work with even more attention and dedication. To quote an aphorism: never give up. You would risk doing it an hour before the miracle!\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? No\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [ { "c_id": "6603", "date": "05 May 2021", "name": "Amado Jiménez-Ruiz", "role": "Author Response", "response": "Dear Dr. Leonardo Varotto, First, we want to thank you for considering our manuscript and the time you and the reviewers devoted to reading it. We have received the feedback and suggestions, and we are addressing them. We have modified the manuscript according to the comments below. Impending paradoxical embolism is an uncommon condition rarely reported in the medical literature. Our pilot exercise for the review concluded that the published information is limited to case reports and small case series and therefore based our data extraction plan and further analysis on these results. During the process of analysis and selection of potential covariables for the project, we considered different methods. Some of those models were Ridge and LASSO. However, when we thought about our problem, we concluded that some variables should be in the model irrespective of their statistical significance (age and sex). In the case of LASSO and its autonomous nature for covariates for model selection, we could not ensure that the model would include both. Also, we performed multiple imputations through MICE; we cannot find a universal method to combine both techniques. For these two reasons, we selected a traditional stepwise approach. Instead of targeting significance for the covariates, we targeted overall accuracy in predictions or AIC. Thanks again for taking the time to review this manuscript. Sincerely, The Authors" } ] } ]	1	https://f1000research.com/articles/9-1437
https://f1000research.com/articles/10-339/v1	04 May 21	{ "type": "Research Article", "title": "Interprofessional diabetes and oral health management: what do primary healthcare professionals think?", "authors": [ "Phyllis Lau", "Anthony Tran", "Matthew Chen", "Evelyn Boyce", "Rachel Martin", "Hanny Calache", "Anthony Tran", "Matthew Chen", "Evelyn Boyce", "Rachel Martin", "Hanny Calache" ], "abstract": "Background: Diabetes and periodontitis have a bi-directional relationship. And yet, collaborations between primary healthcare practitioners in diabetes and oral health care are minimal. This study explored the views of general practice and oral health professionals on the link between diabetes and periodontitis, and interprofessional diabetes and oral health management. Methods: A sequential mixed-methods exploratory research design was used. General practice and oral health professionals were recruited from four community health centres in Melbourne. Quantitative surveys explored participants’ experiences, attitudes and knowledge of diabetes and oral health management and interprofessional collaboration; qualitative follow-up interviews explored survey responses with selected participants. Results: 58 participants completed the online surveys; 22 then participated in semi-structured interviews. Participants generally had strong intentions to collaborate interprofessionally in diabetes and oral health management. Most general practice and oral health professional participants were willing to perform simple screening for periodontitis or diabetes respectively. Themes from the interviews were grouped under three domains: ‘attitude towards diabetes and oral health management’, ‘subjective norms’ and ‘perceived behavioural control’; and an overarching domain to describe participants’ ‘current practice’. Existing siloed primary healthcare practices and lack of formal referral pathways contribute to poor interprofessional collaboration. Most participants were unsure of each other’s responsibilities and roles. Their lack of training in the relationship between general and oral health, compounded by systemic barriers including time constraint, high dental costs, long public dental waiting list and unintegrated health information systems, also impeded interprofessional care. Conclusions: The diabetes and oral health link is not properly recognised or managed collaboratively by relevant primary healthcare professionals in Australia. There is, nonetheless, strong intentions to engage in interprofessional diabetes and oral health care to contribute to improved patient outcomes. Primary healthcare professionals need dedicated and accredited interprofessional training and competencies, formal referral systems and sustainable health policies to facilitate collaboration.", "keywords": [ "interprofessional care", "diabetes mellitus", "oral health", "periodontitis", "primary healthcare", "general practice", "primary dental" ], "content": "Introduction\n\nCurrent evidence shows a bidirectional link between diabetes and chronic periodontal disease (periodontitis). Diabetes is associated with increased risk of an inflammatory response to periodontal micro-biota. Severe periodontitis is three- to four-times more prevalent in people with diabetes. Periodontitis on the other hand seems to affect blood glucose levels in patients with diabetes. Severity of periodontitis may be associated with increased diabetes episodes requiring hospitalisation1–3. People with both diabetes and periodontitis have increased risk of premature tooth loss, poorer diet, poorer diabetes control and more cardiovascular complications. In the 1990s, chronic periodontitis was added as the sixth complication of diabetes mellitus2.\n\nBoth Australian and international guidelines recommend that diabetes care providers should undertake oral health reviews and provide dental referrals if required. The Royal Australian College of General Practitioners (RACGP) recommends to assess the oral health of patients with diabetes4. The International Diabetes Federation (IDF) recommends the strengthening of interdisciplinary collaboration to improve general patient outcomes and as a primary means to prevent periodontitis for patients with diabetes5. Despite these recommendations, the potential for screening patients with diabetes for early management of gum problems is often overlooked in primary care. There are siloes in practice and a lack of collaboration between general practice and oral health professionals6–8. Consistent with current guidelines, general practitioners (GPs) usually prescribe short-term pain relief and/or antibiotics for teeth and gum issues and may advise patients to see a dentist. This is usually the extent of their involvement with oral health management. Similarly, diabetes screening is rarely performed by OHPs despite research showing significant proportions of dental patients have undiagnosed diabetes or pre-diabetes risks3,9,10.\n\nMuch of the current literature nonetheless focuses on assessment of diabetes screening in dental settings or the evolution of the dental profession3,9,11. Some have also explored the views of healthcare professionals on integration of diabetes and periodontitis management including some recent work conducted at the Centre for Oral Health Outcomes, Research Translation and Evaluation at Western Sydney University in New South Wales12–17. However, limited research on interprofessional diabetes and oral health care has been conducted in Victoria or focused on public community health service (CHS) setting.\n\nThis study aimed to explore the knowledge, practice and attitude of Victorian general practice professionals (GPPs) [including GPs, primary health care nurses (PCNs), diabetes educators (DEs)] and oral health professionals (OHPs) [including dentists (Ds), dental hygienists (DHs), oral health therapists (OHTs), dental therapists (DTs) and dental assistants (DAs)] in managing diabetes and periodontitis and their views on interprofessional care in CHS setting.\n\n\nMethods\n\nThis research was approved by human research ethics committees at University of Melbourne (ID 1750835), Deakin University (ID 2018-190) and La Trobe University (ID 1750835).\n\nWritten informed consent from the participants for the publication of findings from this research was obtained. In accordance with the requirements of the ethics committee and the approved research protocol, details that would potentially identify participants due to the small sample size have been removed or replaced with codes in this publication.\n\nThis is a sequential mixed-methods exploratory study. Quantitative online surveys and qualitative semi-structured interviews were conducted with healthcare professionals. Quantitative analysis reported descriptive statistics only. Qualitative analysis used a mixed inductive and deductive approach to explore the experiences of healthcare professionals and reporting was guided by the consolidated criteria for reporting qualitative research checklist (COREQ)18.\n\nAn advisory group guided the implementation of the study. It consisted of representatives from consumers, practitioners (GP, DE, general dentist, periodontist, oral health therapist) and managers of the CHSs involved.\n\nPL is an academic primary care researcher; EB is a diabetes nurse educator; HC is an academic dental public health researcher; MC and AT were honours research students and RM was a public general dentist at the time of the project. The team has an interest in promoting interprofessional primary healthcare.\n\nGPPs and OHPs were recruited purposively from four CHSs in Victoria. The project was presented by the research team to eligible staff at two CHSs; email invitations with a short introduction video were sent to eligible staff via their management teams at the other two. Staff were assured that participation was voluntary. Project description and an anonymous survey link were given to all participants.\n\nOn completion of the survey, participants’ contact details were sought if they opted to participate in follow-up interviews. A matrix (gender, age, professional role) was used to select, for the interviews, a broad representation of those who provided contact details to ensure maximum variation.\n\nTwo online surveys (one for GPPs; one for OHPs) were conducted via the REDCap electronic data capture tool hosted at The University of Melbourne platform19. The questions (Table 1 and Table 2) were developed based on a review of the literature and guided by our advisory group. They were piloted with GP registrars, academic nurses and dentists in the research team’s network before the surveys were rolled out.\n\nLikert scales gauged participants’ agreement with statements relating to confidence, current practice and interprofessional collaboration in oral health or diabetes management and perceived feasibility of screening for periodontitis or diabetes within routine practice. Data were analyzed in Microsoft Excel (2017) (RRID:SCR_016137) to produce descriptive statistics. Google Sheets (RRID:SCR_017679) is a free alternative.\n\nParticipants were asked to opt into interviews to explain their survey responses, identify barriers to diabetes and oral management, and suggest ways to improve interprofessional diabetes and oral health management (Table 3 and Table 4). Author AT interviewed GPPs whilst author MC interviewed OHPs either by phone or in-person at the participants’ practice. Both were trained by author PL in interview techniques and did practice interviews with authors PL and HC. Questions were pilot tested with students in the Department of General Practice Honours student cohort prior to conducting the interviews.\n\nInterviews were audio-recorded and transcribed, and field notes were taken. Transcripts were offered to participants for review before being imported into QSR International's NVivo 12 qualitative analysis software (RRID:SCR_014802)20. RQDA package for R (RRID:SCR_001905) is an open-source alternative. AT coded all GPP interviews, MC coded all OHP interviews while the rest of the team (PL, EB, RM and HC) coded up to six interviews each, ensuring every transcript was coded by at least two researchers. Transcripts were first inductively coded separately and then collectively by the research team. Following several iterative meetings to reach consensus in coding and categorising differences, the research teams decides that the Theory of Planned Behavior model (TPB) which outlines three domains affecting intention to perform a behavior: attitude towards the behavior (or beliefs which influence an individual to perform a behavior), subjective norms (or perceived external pressures as influenced by judgement of others) and perceived behavioral control (or ease or difficulty in performing the behavior as determined by external factors) is congruent with patterns emerging21. Deductive analysis using a framework analysis approach then followed using the TPB to identify patterns and elicit themes22. The team continued to meet to discuss the themes elicited until agreement was reached.\n\n\nResults\n\nA total of 58 participants completed the survey between April and July 2018: 20 from general practice (eight GPs, nine PCNs and three DEs) and 38 from dental practice (18 Ds, four DHs, six OHTs, five DTs and five DAs). (Table 5)\n\nNote: n/a = question not asked.\n\nTable 6 shows the survey results. Most GPPs (75%) had no oral health training in their professional education. The majority rarely or never assessed the mouths of patients (70%) and were not confident in identifying oral disease (60%), discussing oral health with their patients (55%) or managing oral health in patients with diabetes (80%).\n\nIn contrast, most OHPs (74%) learnt the relationship between oral health and diabetes in their professional training. The majority were confident in identifying risk factors of type 2 diabetes (66%) and discussing diabetes with their patients (82%) and managing patients with both diabetes and periodontal disease (82%). However, most rarely or never consult GPs (69%). Most GPPs (55%) occasionally referred patients to OHPs while most OHPs rarely or never referred patients to GPPs.\n\nAll GPPs agreed that oral health screening was within their role (100%) and most were comfortable to perform simple oral health screening (80%). All thought that oral health screening was feasible in practice (100%) but most thought that it would be welcome by their colleagues (80%). Almost all (95%) welcomed oral health training specifically in diabetes management. All agreed that OHPs should screen patients with periodontitis for diabetes (100%) and almost all thought that better interprofessional collaboration would benefit patients (95%). These results are similar to those from corresponding statements for the OHPs. However, only 65% of GPPs said they would welcome the opportunity for continuing education/training in oral health, compared with 95% of OHPs who said they would welcome continuing education/training in diabetes.\n\nFive GPPs (four PCNs, three DEs) and 10 OHPs (four Ds, two DHs, two OHTs, one DT and one DA) were further interviewed. Interviews lasted 20 minutes on average. One participant declined to be audio-recorded; none took up the offer to review their transcripts or offer additional feedback.\n\nData saturation was determined to have been reached. Ten themes were grouped under the three TPB domains and an additional overarching domain to describe participants’ current practice.\n\nDomain 1: current practice\n\nTheme 1: separate diabetes and oral health management\n\nMost GPP acknowledged that they did not routinely assess the mouth of their patients with diabetes.\n\n“I don’t usually do it routinely unless there is a particular symptom that they complained of or as I am talking to them I can see that they have got an oral health issue” GP2, female, 51–60 years old, worked 11–20 hours per week, 25–30 years’ experience\n\nOHPs on the other hand often discussed the diabetes and oral health link during initial patient examination.\n\n“…if the patient says they have diabetes or has maternal or paternal history of diabetes, I discuss with my patients the risk he and she can have. If he or she has already been diagnosed with gum disease, I inform them about why it’s so important that (their diabetes) should be controlled.” D2, female, 31–40 years old, worked 31–40 hours per week, 6–10 years experience\n\nTheme 2: poor interprofessional communication or collaboration\n\nEven where medical and dental services were co-located, they were siloed in practice.\n\n“I have dentists on-site here, but we only really get called when someone is feeling faint. There is little two-way communication.” GP5, female, 51–60 years old, worked 21–30 hours per week, 25–30 years’ experience\n\nTheme 3: lack of formal referral process\n\nMost participants tended to refer patients to each other informally.\n\n“I would just ask them if they have seen the dentist. Then they would say yes or no. If they haven’t then I would urge them to go (and) make an appointment with the dentist.” GP2, female, 51–60 years old, worked 11–20 hours per week, 25–30 years’ experience\n\n“So I haven’t referred any patients to a GP directly to get it (diabetes) screened, but I have requested them to see a GP to make sure that their diabetes is under control so I can go ahead with my treatment plan.” D2, female, 31–40 years old, worked 31–40 hours per week, 6–10 years’ experience\n\nGPPs noted that they received little feedback from OHPs following ‘referral’.\n\n“When I refer patients to a physiotherapist or a psychologist, or a cardiologist, I get a letter back. I don’t get anything back from our dental services.” GP1, male, >60 years old, worked 21–30 hours per week, >30 years’ experience\n\nFormal referrals from OHPs to non-GP health professionals were more common.\n\n“I have never referred to a GP for diabetes. We do have diabetes educators… and I would refer for that.” D1, female, 31–40 years old, worked 31–40 hours per week, 11–15 years’ experience\n\nDomain 2: attitude towards diabetes and oral health management\n\nTheme 4: responsibilities and roles\n\nMany GPPs admitted that oral health was generally overlooked. Many did not think oral health should be their responsibility.\n\n“I don’t think we really know what to do, I think we really leave that to our dental colleagues” GP3, male, 31–40 years old, worked 31–40 hours per week, <5 years’ experience\n\nIn contrast, most OHPs thought they should have a role in diabetes screening.\n\n“I think it should be (within our responsibilities). It isn’t though, at the moment.” OHT2, female, <30 years old, worked 31–40 hours per week, <5 years’ experience\n\nHowever, two dentists expressed apprehension about the ‘unfamiliar territory’ of the Australian Diabetes Risk Assessment (AUSDRISK) tool.\n\n“Another thing is the waist measurement. I don’t know about that. It’s also not really in our place to do so.” D4, female, <30 years old, worked >40 hours per week, <5 years’ experience\n\nGPPs generally agreed that diabetes risk screening is viable in the dental setting.\n\n“…(screening) for diabetes is so simple these days it doesn’t even require a fasting blood test, let alone a glucose tolerance test” GP1, male, >60 years old, worked 21–30 hours per week, >30 years’ experience\n\nMost OHPs also felt GPs and nurses could conduct simple oral health screening and prevention. However, some opposed the idea.\n\n“No, I don’t think (non-dental practitioners should look in patients’ mouth). A doctor can, in a general way. But I don’t think they can make a diagnosis about what the problem is...” DH1, male, 31–40 years old, worked 21–30 hours per week, <5 years’ experience\n\nTheme 5: further training\n\nAlmost all participants felt further training was needed to improve confidence and competence. However, it needs to be conducive for healthcare professionals to attend.\n\n“But it would need to come out of my paid clinical time and have CPD (continuing professional development) points.” D3, female, 31–40 years old, worked 31–40 hours per week, 6–10 years’ experience\n\nSeveral participants commented on the value of interdisciplinary education.\n\n“Probably doing things like professional development together, you know, once a year or something like that. That would certainly increase my knowledge… It would also begin to build those working relationships” GP5, female, 51–60 years old, worked 21–30 hours per week, 25–30 years’ experience\n\nTheme 6: interprofessional collaboration\n\nOverall, participants recognized the benefits of interprofessional collaboration.\n\n“It shows that we’re creating a united front on the importance of it, and we are taking it seriously and working in collaboration to improve the health of the clients.” OHT1, female, 31–40 years old, worked 21–30 hours per week, 11–15 years’ experience\n\nMany participants however were hesitant about involving time-poor GPs and dentists.\n\n“Yeah, especially between nurses and dental nurses we can be involved. But leave doctors and dentists if they are so busy…” PCN2, female, 41–50 years old, worked 21–30 hours per week, <5 years’ experience\n\nDomain 3: subjective norms\n\nTheme 7: patients’ knowledge and priority of oral health\n\nParticipants thought patients were generally unaware of the relationship between diabetes and oral health.\n\n“Clients are not hugely aware (of the) link of oral health and diabetes, and the bi-directional link…” – OHT1, female, 31–40 years old, worked 21–30 hours per week, 11–15 years’ experience\n\nSome OHPs said that patients did not appreciate the need to discuss diabetes with them…\n\n“There have been a couple of patients who didn’t want to discuss diabetes.” – DH1, male, 31–40 years old, worked 21–30 hours per week, <5 years’ experience\n\n…or prioritized oral health.\n\n“The teeth are the last thing that’s important to them.” – DT1, female, 31–40 years old, worked 21–30 hours per week, 16–20 years’ experience\n\nTheme 8: perceived resistance from colleagues to change scope of practice\n\nMany GPPs did not think their fellow colleagues would accept oral health as part of their responsibilities.\n\n“I discussed this with my colleagues just recently, a lot of us believe it’s not really within our scope, and we are not going to venture into an area that we are not that familiar with” DE3, female, 31–40 years old, worked 21–30 hours per week, <5 years’ experience\n\nMany participants contended that the culture of siloes was a barrier.\n\n“I think it’s just the way the (health) profession has been for so long. Each person just does their own thing, and there’s no collaboration.” DT1, female, 31–40 years old, worked 21–30 hours per week, 16–20 years’ experience\n\nDomain 4: perceived behavioral control\n\nTheme 9: lack of opportunity for training\n\nParticipants highlighted a lack of opportunities for further training.\n\n“I have had absolutely no training on dental health apart from growing up in a family where we were trained to brush our teeth” – GP5, female, 51–60 years old, worked 21–30 hours per week, 25–30 years’ experience\n\nMany participants perceived that their availability for training was in fact not within their control.\n\n“It depends on my manager… how much she can provide us with the training hours.” PCN2, female, 41–50 years old, worked 21–30 hours per week, <5 years’ experience\n\nTheme 10: systemic barriers\n\nTime constraint was a barrier for almost all participants.\n\n“Time is a huge issue. I have mostly half-an-hour appointments, which is a very limited scope for me because I have other things to do as well… To include everything in that half an hour would be very tough and a bit of a problem.” DH1, male, 31–40 years old, worked 21–30 hours per week, <5 years’ experience\n\nThis excuse, however, was quashed by other participants.\n\n“It doesn’t take that long to do and we can do it. I have been listening to people say that “We don’t have time to do it”, but I think that we can just make time. It’s an important thing to do.” OHT1, female, 31–40 years old, worked 21–30 hours per week, 11–15 years’ experience\n\nSome participants thought that the lack of software uniformity and integration of information technology between professions hampered collaboration.\n\n“Dental files are dental files and medical files are medical files. … the only person you’re relying on is what the patient relays back to you, and sometimes they don’t even know what’s being told to them except use this medication, get your dental check-up on this day.” – DT1, female, 31–40 years old, worked 21–30 hours per week, 16–20 years’ experience\n\nHigh dental costs and long public dental waiting list were the most common reasons that GPs, PCNs and DEs gave for their reluctance to refer patients to OHPs.\n\n“Another barrier is cost… (Patients) are so used to bulk-billing and they thought that if medical bulk bills, why not dental as well.” PCN2, female, 41–50 years old, worked 21–30 hours per week, <5 years’ experience\n\n“…even the minor delay of even a week or two is sufficient for the patient to scurry away and say I’ll do it another time, and then the opportunity is lost.” GP1, male, >60 years old, worked 21–30 hours per week, >30 years’ experience\n\n\nDiscussion\n\nOur research aims align with the National Oral Health Plan’s recommendations for greater collaboration of OHPs with the broader health workforce23. Our findings contribute to a growing evidence base for interprofessional collaboration between medical and oral health professionals and will help support the RACGP guidelines on diabetes management and IDF guidelines on interprofessional collaboration9,10. This corresponds with the recommendations from a UK study to develop initiatives and policies to promote and embed oral health management as part of diabetes care24.\n\nThe TPB model provided the framework to explain the key factors influencing healthcare professionals’ consideration of interprofessional care of diabetes and periodontitis21. Several attitudinal beliefs and societal normative influences strongly impact their collaborative behavioral patterns. Our results are similar to those from studies that have found many non-oral healthcare professionals do not manage the oral health of patients with diabetes25,26.\n\nLike our study, a German study also reported a lack of collaboration from OHPs which was likely a result of the informal nature of ‘verbal referrals’ usually directed at OHPs27. Other research shows that OHPs supported diabetes screening becoming part of oral health professionals’ standard care but the convoluted referral system dissuaded them from providing formal referrals8. It is important that a simple and structured referral system, like the one between medical specialists, be developed between medical/nursing practitioners and OHPs to promote effective interprofessional collaboration.\n\nCurrently, Australian medical and dental practices use completely different information systems that are not integrated. This compounds service fragmentation and suboptimal clinical outcomes. Appropriate policies are required to incorporate information sharing in health systems to support interprofessional collaborative relationships28.\n\nIt was not surprising that time constraint was a barrier particularly for GPs and dentists. They may be more suited to be involved after the initial primary prevention strategies. The barrier of the healthcare profession ‘silo’ culture is well-known and is also reflected in Marshall and Spencer’s paper which cites a “separateness” between Australian medical and dental practices29. However, improved management of periodontitis would potentially improve blood glucose control, which would in turn further improve periodontal health resulting in longer-term fewer visits to GP and dental clinics and ultimately save time and resources.\n\nFurther training in diabetes and oral health management would increase healthcare professionals’ knowledge and confidence30. Ward et al. found that nurses who were confident with their oral health education were more likely to screen patients with diabetes for periodontitis25. The importance of interdisciplinary training is consistent with Lamster and Eaves’ push for greater interprofessional collaboration and emphasis on respecting all health disciplines, increasing the understanding of each profession’s role, providing more effective communication and maximizing safety, efficiency and effectiveness11. Currently there are minimal interprofessional training opportunities. Development of future training should have an interprofessional focus, be as conducive as possible and be accredited for CPD.\n\nOur mixed-methods approach allowed an in-depth exploration of participants’ views. Although the sample size was small, the wide range of healthcare professionals provided broad perspectives. Unequal representation from different professional groups may impede the generalizability of the findings even though data saturation was reached. Our focus on CHSs with co-located general practice and dental services may have limited the extrapolation of our findings to other settings.\n\n\nConclusion\n\nPrimary healthcare professionals generally recognized the importance and have strong intentions to engage in interprofessional diabetes and oral health management. Accredited interprofessional training should bridge the divide between medicine and dentistry. Formal referral processes are necessary to improve interprofessional feedback and communication. Health policies and advocacies need to target dental costs and public dental waiting lists to motivate referrals. An effective and feasible interprofessional collaborative diabetes and oral health care model would contribute to improved patient outcomes. Future studies should include the views of patients, policy makers and other stakeholders.\n\n\nData availability\n\nDeidentified data of this research will only be provided on request. Reviewers or other researchers intending to reproduce the study may make this request by emailing the corresponding author. This conditional withholding of data is necessary to protect the privacy and confidentiality of the participants who were sourced from a small number of community health services and the final sample size was small.\n\nFigshare: COREQ checklist for ‘Interprofessional diabetes and oral health management: what do primary healthcare professionals think?’ https://doi.org/10.26188/14454372.\n\nData are available under the terms of the Creative Commons Attribution NoDerivatives 4.0 International license (CC-BY-ND 4.0).", "appendix": "Author contributions\n\n\n\nPhyllis Lau (PL), Hanny Calache (HC) and Rachel Martin (RM) contributed to the conceptualisation and design of the study, funding acquisition, recruitment of participants, collection of the raw data and data analysis. Evelyn Boyce (EB) managed the project administration and assisted with recruitment of participants, data collection and analysis. Anthony Tran (AT) and Matthew Chen (MC) were Honours research students at the time and, under the supervision of PL and HC, conducted the surveys and interviews, and analysed the data as part of the research team. All authors contributed to the drafting, revisions and final approval of the version to be published.\n\n\nAcknowledgements\n\nThe authors acknowledged the contributions of Prof Mark Gussy, Assoc Prof John Furler and our advisory group who guided the progress of the research, the staff at the four community health services who assisted with recruitment of participants and the participants who contributed their valuable time and views in the surveys and interviews.\n\n\nReferences\n\nPreshaw PM, Alba AL, Herrera D, et al.: Periodontitis and diabetes: a two-way relationship. Diabetologia. 2012; 55(1): 21–31. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLoe H: Periodontal disease. The sixth complication of diabetes mellitus. Diabetes Care. 1993; 16(1): 329–34. PubMed Abstract \| Publisher Full Text\n\nLalla E, Kunzel C, Burkett S, et al.: Identification of unrecognized diabetes and pre-diabetes in a dental setting. [Erratum appears in J Dent Res. 2012 Jul; 91(7): 715] J Dent Res. 2011; 90(7): 855–60. PubMed Abstract \| Publisher Full Text\n\nRoyal Australian College of General Practitioners (RACGP) and Diabetes Australia: General practice management of type 2 diabetes – 2014– 15. 2014. Reference Source\n\nIDF Clinical Guidelines Task Force: IDF Guideline on oral health for people with diabetes. Brussels: International Diabetes Federation, 2009. Reference Source\n\nWright D, Muirhead V, Weston-Price S, et al.: Type 2 diabetes risk screening in dental practice settings: a pilot study. Br Dent J. 2014; 216(7): E15. PubMed Abstract \| Publisher Full Text\n\nMarshall S, Schrimshaw EW, Metcalf SS, et al.: Evidence from ElderSmile for diabetes and hypertension screening in oral health programs. J Calif Dent Assoc. 2015; 43(7): 379–87. PubMed Abstract \| Free Full Text\n\nGlurich I, Schwei KM, Lindberg S, et al.: Integrating medical-dental care for diabetic patients: qualitative assessment of provider perspectives. Health Promot Pract. 2017: 19(4): 531–541. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGenco RJ, Schifferle RE, Dunford RG, et al.: Screening for diabetes mellitus in dental practices: a field trial. J Am Dent Assoc. 2014; 145(1): 57–64. PubMed Abstract \| Publisher Full Text\n\nFranck SD, Stolberg RL, Bilich LA, et al.: Point-of-care HbA1c screening predicts diabetic status of dental patients. J Dent Hyg. 2014; 88(1): 42–52. PubMed Abstract\n\nLamster IB, Eaves K: A model for dental practice in the 21st century. Am J Public Health. 2011; 101(10): 1825–30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAl-Habashneh R, Barghout N, Humbert L, et al.: Diabetes and oral health: doctors' knowledge, perception and practices. J Eval Clin Pract. 2010; 16(5): 976–980. PubMed Abstract \| Publisher Full Text\n\nTse SY: Diabetes mellitus and periodontal disease: awareness and practice among doctors working in public general out-patient clinics in Kowloon West Cluster of Hong Kong. BMC Fam Pract. 2018; 19(1): 199. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nObulareddy VT, Nagarakanti S, Chava VK: Knowledge, attitudes, and practice behaviors of medical specialists for the relationship between diabetes and periodontal disease: A questionnaire survey. J Family Med Prim Care. 2018; 7(1): 175–178. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYuen HK, Onicescu G, Hill EG, et al.: A survey of oral health education provided by certified diabetes educators. Diabetes Res Clin Pract. 2010; 88(1): 48–55. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPoudel P, Griffiths R, Wong V, et al.: Perceptions and Practices of Diabetes Educators in Providing Oral Health Care: A Qualitative Study. Diabetes Educ. 2018; 44(5): 454–64. PubMed Abstract \| Publisher Full Text\n\nPoudel P, Griffiths R, Wong V, et al.: Perceptions and practices of general practitioners on providing oral health care to people with diabetes - A qualitative study. BMC Fam Pract. 2020; 21(1): 34. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTong A, Sainsbury P, Craig J: Consolidated criteria for reporting qualitative research (COREQ): a 32-item checklist for interviews and focus groups. Int J Qual Health Care. 2007; 19(6): 349–57. PubMed Abstract \| Publisher Full Text\n\nMelbourne Clinical and Translational Science: RedCap: research electronic data capture. Melbourne: The University of Melbourne; 2020.\n\nRichards L: Handling qualitative data: a practical guide. London: Sage Publications; 2005. Reference Source\n\nAjzen I: The theory of planned behavior. Organ Behav Hum Decis Process. 1991; 50(2): 179–211. Publisher Full Text\n\nGale NK, Heath G, Cameron E, et al.: Using the framework method for the analysis of qualitative data in multi-disciplinary health research. BMC Med Res Methodol. 2013; 13(1): 117. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAustralian Government: Australian's National Oral Health Plan 2015-2024: Healthy Mouths Healthy Lives. Adelaide: Coalition of Australian Governments Health Council, 2015. Reference Source\n\nBissett SM, Stone KM, Rapley T, et al.: An exploratory qualitative interview study about collaboration between medicine and dentistry in relation to diabetes management. BMJ Open. 2013; 3(2): e002192. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWard AS, Cobb CM, Kelly PJ, et al.: Application of the theory of planned behavior to nurse practitioners' understanding of the periodontal disease-systemic link. J Periodontol. 2010; 81(12): 1805–13. PubMed Abstract \| Publisher Full Text\n\nLopes MH, Southerland JH, Buse JB, et al.: Diabetes educators' knowledge, opinions and behaviors regarding periodontal disease and diabetes. J Dent Hyg. 2012; 86(2): 82–90. PubMed Abstract\n\nHolzinger F, Dahlendorf L, Heintze C: 'Parallel universes'? The interface between GPs and dentists in primary care: a qualitative study. Fam Pract. 2016; 33(5): 557–61. PubMed Abstract \| Publisher Full Text\n\nSteihaug S, Johannessen A, Ådnanes M, et al.: Challenges in achieving collaboration in clinical practice: the case of Norwegian health care. Int J Integr Care. 2016; 16(3): 3. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMarshall R, Spencer A: Accessing oral health care in Australia. Med J Aust. 2006; 185(2). PubMed Abstract \| Publisher Full Text\n\nAl-Khabbaz AK, Al-Shammari KF, Al-Saleh NA: Knowledge about the association between periodontal diseases and diabetes mellitus: contrasting dentists and physicians. J Periodontol. 2011; 82(3): 360–6. PubMed Abstract \| Publisher Full Text" }	[ { "id": "84496", "date": "18 May 2021", "name": "Judith Haber", "expertise": [ "Reviewer Expertise Relationship of Oral Health to Overall Health" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well-designed mixed methods study that is based on a current review of the literature. The focus of the study highlights a significant global population health problem, diabetes and periodontal disease and seeks to identify the potential barriers and facilitators of effective interprofessional care for this population. The surveys and interview guides are well constructed, the data analysis appropriate, and the findings are reported in a very understandable way that elucidates their potential applicability in education and practice.\nI would accept this well written manuscript and am confident that it will be an important contribution to the literature both in Australia and the United States.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6686", "date": "20 May 2021", "name": "Phyllis Lau", "role": "Author Response", "response": "We thank the reviewer for her very positive and encouraging comments. Much appreciated!" } ] }, { "id": "98065", "date": "02 Dec 2021", "name": "Shahida Mohd-Said", "expertise": [ "Reviewer Expertise Oral Health Management Innovation Research: Teledentistry and Integrated Care Management of Periodontitis Patients" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper describes the oral health care professionals' qualitative input on their personal experiences, knowledge, attitude, and interprofessional collaboration when managing diabetes in Melbourne, Australia. The aim of the study is relevant and critical, and generally, it was conducted using a comprehensive, relevant, good, and clear methodology as well described in the paper. Perhaps the inclusion of more references within 2018-2021 would benefit this paper since more closely relevant qualitative studies have been published recently involving health professionals and concepts in interprofessional or shared care for patients with diabetes.\nRevision to the main conclusion would be beneficial to reflect the study findings more accurately rather than generalising the statement to all primary healthcare professionals.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-339
https://f1000research.com/articles/10-338/v1	04 May 21	{ "type": "Research Article", "title": "The impact of Indonesian oil price (CPI) and macroeconomics on investments in the manufacturing sector in Indonesia", "authors": [ "Handri Handri", "Hendrati Dwi Mulyaningsih", "Achmad Kemal Hidayat", "Rudi Kurniawan", "Ani Wahyu Rachmawati", "Handri Handri", "Achmad Kemal Hidayat", "Rudi Kurniawan" ], "abstract": "Background: Indonesia consumes oil as the main energy source in the production process and as a result of the development of the manufacturing industry. Thus, investment in manufacturing stocks will be affected by oil price fluctuations and macroeconomic conditions. Changes in oil prices will affect the performance of the manufacturing sector which in turn affects manufacturing stock prices. This paper aims to examine the impact of Indonesia's oil price shocks and macroeconomic factors on stock price movements in the manufacturing sector. Methods: This study uses monthly data for the 2009-2016 period in the manufacturing sector, and 67 stocks were selected on the basis consistently available in the period of the research. The cointegration and causality technique was used in this paper; firstly we applied a unit-panel root test, Secondly, we performed a residual test to indicate whether there was cointegration among variables in the long run equilibrium, and short the short run, we used a Granger causality test. Results: The panel unit root test (both Shin and Fisher) and the Pedroni cointegration residual test show that the data is stationary at 1% level of significance, thus all variables simultaneously achieve long-run equilibrium, and in the short run, the Granger causality test shows that there is one way direction causality Conclusions: For long-term investment in manufacturing stocks, investors must consider the exchange rate, as it is also as a determining factor in influencing the movement of manufacturing stock prices, inflation, and the production index. Meanwhile, weakening of the rupiah in the short run will also determine investment conditions due to the dependency on raw materials for production from foreign sources. The price of oil as an energy source in the manufacturing sector does not have a long-term relationship with other variables.", "keywords": [ "Oil price (CPI)", "stock price", "macroeconomics", "cointegration", "causality" ], "content": "Introduction\n\nOil is probably the most important source of energy in the economy, and also an important component of the production cost of companies in the manufacturing sector. Fluctuations in oil prices will therefore affect the performance of the manufacturing sector. Disruption of the sector's performance will also cause fluctuations in the share price of the manufacturing sector. Furthermore, as far as we know, stock price movements are influenced by internal and external factors caused by macroeconomic conditions. However, investors can still invest even in unstable macroeconomic conditions, but need to be more prudent.\n\nAs a developing country, the manufacturing sector in Indonesia has an important role in producing goods to meet the needs of a growing market. To attract investors, macroeconomic conditions need to be the focus of companies and investors in developing investments. The macroeconomic conditions which were chosen as the focus of this study were inflation, the exchange rate of the rupiah against the dollar, the index of large and medium production and fluctuations in Indonesian oil prices (CPI).\n\nChanges in oil prices are important because they have implications for stocks as a whole and in general are a portfolio strategy. This information is very important for investor strategies, so that investors are still able to get a return on investment by minimizing risk while oil prices are still uncertain.\n\nThe results of the study, which show the causality and cointegration relationship of oil price shock and macroeconomics to the dynamics of manufacturing sector stock price fluctuations, can be a long- and short-term consideration for companies and investors. It can also be a guideline for companies in developing production and a consideration for investors.\n\nTheoretically, there are various reasons for which oil price can affect stock prices (Cai et al., 2017; Hamilton, 2008; Jones & Kaul, 1996; Rafailidis & Katrakilidis, 2014; Ramos & Veiga, 2013). One of the reasons relates to the impact of changes in oil price on the future expected discounted earnings of companies. An increase in oil price will increase the cost of production which in turn will reduce the profit and the future expected discounted earnings and, therefore, the stock prices. Another reason is related to the effect of changes in the stock prices of oil producing companies on the stock prices of other companies whose expected future earnings flows are adversely affected by the increase in oil prices. An increase in oil price will be beneficial for oil producing companies and will increase their stock prices. However, this puts pressure on the stock prices of other non-oil producing companies. Moreover, increased oil prices may create uncertainty for the economy and also for non-oil companies, which may result in delayed investment decisions which will adversely affect their stock prices.\n\n\nMethods\n\nOur dataset is monthly, beginning with January 2009 and ending with December 2016, a relatively rich sample sufficient for conducting the type of econometric modeling in this paper. Like most previous studies on the same topic, this study employs variables that include oil prices, stock prices, real output, inflation and exchange rates. As the proxy for the oil prices we use the CPI index, which is an average price of a basket of internationally traded Indonesian crude oils that is still used in some contracts today. The proxy for the manufacturing sector stock prices is the manufacturing sector closing price index obtained from the Indonesian Stock Exchange. For the output, because data on real output or real GDP are only available quarterly, then the industrial production index, which is available monthly, is used as a proxy. Inflation is calculated as the percentage change of the consumer price index.\n\nTo investigate the impact of changes in oil price on manufacturing sector returns, we perform cointegration and Granger causality analysis. Prior to both analysis it is necessary to investigate the stationarity properties of panel data by performing unit root tests. The software used in calculating the econometric model of the study is eviews10.\n\nThe first test of the data is carried out using the unit root test to determine whether the data is stationary at the level, this is the unit root test which is performed to avoid spurious regression results. The unit-panel root test method used in this study consisted of the Im, Pesaran and Shin methods together with the Fisher method. In the latest literature, the panel-based unit root test has higher strength than the time series-based unit root test.\n\nIt is important to check the stationarity of data before proceeding with further analysis because often a method designed for stationary series is not suitable for non-stationary series, and if applied one may run the risk of obtaining completely misleading results (Nason, 2013). We check the stationarity of the data involved by employing the Im, Pesaran, and Shin (IPS) unit root test which is based on the following equation:\n\n\n\nWhere ∆y(i,t) is the first difference of the dependent variable, βi is the coefficient of the lagged of the dependent variable. The null hypothesis of the test is all series follow a unit root process: βi = 0 for all i, against the alternative that some (but not all) have unit roots. Rejecting the null implies that series in the panel are stationary.\n\nAs an alternative approach to panel unit root tests, we also use Fisher’s (1932) results to derive tests that combine the p-values from individual unit root tests. This idea has been proposed by Maddala & Wu (1999), Choi (2001) and Hurlin (2010). The null and alternative hypotheses are the same as IPS.\n\nTo check whether a cointegrating, or long-run, relationship exists among variables, the methodology introduced by Pedroni (1999) is employed. This is a residual based test that includes several tests that allow heterogeneity of error variance and deterministic trend across panel members. Hence, the first step is to obtain the residuals from the following regression for each of the panel member:\n\n\n\nWhere t = 1, … , T; i = 1, … , N; m = 1, … , M, where M is the number of regressors; εi,t are residuals that indicate deviatios from the long-run equilibrium. Variables y and x are assumed to be integrated of order one, or I(1). In the test equation, intercept α, coefficient of time trend and slope coefficients β are allowed to vary across panel individuals. The following step is to pool residuals from the previous stage and test for their order of integration by estimating following equation:\n\n\n\n\n\nThe null hypothesis of the test is that ρi = 0 for all i, i.e. there is no cointegration relationship. The alternative hypothesis is that ρi < 0 for i = 1, … , N and ρi = 0 for i = N + 1, … , N.\n\nA variable is said to be Granger-caused by another variable if including the second variable in the information set will improve the forecast of the first variable. The validity of causal test is conditional upon testing the unit root and cointegration among the variables. To undertake the Granger causality test, we consider a k-variate panel vector Autoregression (pVAR) model as follows:\n\n\n\nWhere Yi,t is (1 × k) a vector of variables of interest, A1, A2, … , Ap are (k × k) matrices of parameters to be estimated, ui and ei,t are (1 × k) vectors of dependent variables-specific panel fixed-effects and idiosyncratic error terms, respectively. The order of the model can be determined according to Schwarz’s Bayesian information criterion (BIC) which is one of the most successful criteria.\n\nPairwise Granger causality (Table 1) is a short-term variable relationship test conducted jointly, and the null hypothesis states that there is no joint causality relationship. The causality relationship is found in the second equation where y is not Granger to the variable x. If, statistically, the x and y coefficients are not significant, then there is another relationship between x and y. While if there is a one-way relationship from x to y, then the estimation coefficient is at lag y. Conversely, the one-way relationship from y to x is an indication of the estimated coefficient on lag x.\n\nSource: compiled from various sources.\n\nStacked causality test (typical coefficient) panel data. The short-term relationship in this study uses the Granger causality pairwise test, the first step taken is the stacked causality test approach (Dumitrescu & Hurlin, 2012), where the causality is stacked in a panel data set using the assumption of the same coefficient in all sections with the following equation:\n\n\n\nDumitrescu-Hurlin (heterogeneous or unequal coefficients) panel causality test. The second approach uses heterogeneous coefficients with the Dumitrescu-Hurlin method, in this method, all coefficients are different, in other words, there is transverse heterogeneity with the following equation (Dumitrescu & Hurlin, 2012):\n\n\n\n\nResults\n\nBased on descriptive statistics, all variables have varying averages and standard deviations. The monthly average of stock prices of the manufacturing sector moves following a parabolic pattern, and ranges from 135 to 307.85 during the study period. The sub period from 2009 to 2013 has the highest standard deviation with an average of 65.63. The maximum standard deviation of 288.3 is reached in 2014.\n\nThe oil price index shows an increasing trend with a maximum of 238.63 in 2013. The standard deviation of the oil price index fluctuates within the range from 29.71 in 2009 to the highest of 34.33 in 2014, which suggests a quite large fluctuation in the oil price.\n\nThe consumer price index shows an increasing trend until 2012 with an average of 141.66, and after that follows a decreasing trend with an average of 185.2. The standard deviation during the study period fluctuates within the range from 21.26 to 29.71. The monthly production index fluctuates, with a tendency to decrease in 2009 with monthly average of 103.7, decreasing further in 2010 with a monthly average of 80.5, and then continually increasing to reach a monthly average of 105.1 in 2016. The exchange rate index fluctuates within a range from 81.78 to 119.74, which is a quite large.\n\nHaving examined the descriptive statistics, we proceed to test for the stationarity properties of the data relying on the IPS and Fisher panel unit root test. The results are presented in Table 2 and as can be seen in the table, at the 1% significance level, both the IPS and Fisher unit root tests provide strong evidence that the variables under study are unit-root variables of order one. In other words, they are integrated of order one.\n\nInformation: significance 1%*, significance 5%, significance 10%.\n\nStationary data are an important factor for getting estimates in the long run to avoid false regression results. The unit root test is performed at the beginning before estimation is done to find out whether the data has been stationary at the level or not.\n\nBased on panel unit root statistics we proceed to panel cointegration analysis in order to examine long-run relationship among the variables. The first stage is to determine that the relationships among variable are not spurious, the panel unit root result should be tested by the Fisher and Shin method in stationary at level. The result of the panel unit root test can be seen in Table 2 below.\n\nThe results of the Lm test, Shin and Fisher for all variables in the table above show that the data is stationary at the significance level of 1%.\n\nThe objective of the cointegration test is to test the long-term relationship between oil price shocks, macroeconomics, and stock prices in the manufacturing sector. The cointegration test uses the Pedroni method, where the dimensions are grouped into two types, namely dimensions based on \"within dimensions \" and dimensions \"between dimensions \". Dimension testing \"within dimensions \" is carried out on the respective values of panel v stat, panel rho stat, panel PP stat and panel adf stat. Meanwhile, the \"between- dimensions \" dimension test was carried out on the group rho stat, group PP stat and group ADF stat.\n\nThe relationship of variables in the panel data is of concern at this time. With the cointegration of the Pedroni method, it is known that all variables in this study are co-integrated in the long run, meaning that all the variables used have linear dependence (cointegration), where all variables are cointegrated to achieve mutual balance together in the long run. All the results of the test for both \"within dimensions” and \"between- dimensions \" are shown in Table 3 below.\n\nInformation: significance 1%, significance 5%, significance 10%*.\n\nIn Table 3, the Pedroni cointegration residual test indicates cointegration with a significance level of 1%, so we reject the null hypothesis that there is no cointegration. This means that variable oil price shocks, macroeconomics, and stock prices in the manufacturing sector achieve long-run equilibrium.\n\nFor the short run we use pairwise granger causality to see the relationship in the short term, whether there is a two-way center, one-way causality, or no causality at all, the result of Pairwise granger causality test can be seen in Table 4 below.\n\nTable 4 shows that there is no bidirectional causality, and only one directional causality relation between variables. One-way direction relation is shown between the production index and inflation, and between inflation and both the stock price and exchange rate,\n\nIt can be seen how much influence the cointegration of each variable has on other variables, with further testing the relationships between variables in this study are shown in Table 5.\n\n\nDiscussion\n\nFrom the results, it can be seen from the exchange rate, inflation and the joint production index, that the price of oil is co-integrated in the long run. Individually, it is known that manufacturing shares are integrated with oil prices and exchange rates. Furthermore, oil prices are significantly related to production and inflation indexes. This condition illustrates that, if the exchange rate cannot be maintained in stable conditions and is accompanied by high inflation, the shock of oil prices will affect the company's production activities in the long run.\n\nThe exchange rate of the rupiah will affect manufacturing production due to the large dependence on foreign raw materials to meet production goals in achieving long-term production balance. For long-term investment, it is very important to see changes in the exchange rate and the medium to large production index because these have a two-way causality and a significant positive effect in the short term. If the rupiah strengthens against the US dollar and this continues in the future, it will have an impact on rising production indices in the short term and this condition will also raise the production index in the long run, and vice versa.\n\nWhile the increased purchasing power of consumers, in addition to reflecting the increased ability to invest in the community, also has a positive effect on the ability of the public to invest in manufacturing stocks, an increase in production will trigger an increase in oil prices.\n\nFurthermore, the significant relationship of oil in the short term is based on the W-stat, but on the other hand, based on the Zbar-stat the price of oil is only related to the production index. It can be interpreted that the price movement of oil is divided due to the supply of oil producers and the demand of producers as consumers who use oil.\n\n\nConclusions\n\nWhen people's purchasing power in the short term has a positive effect on investment, in the long run, this condition will strengthen long term investments, so that investment managers can hope to improve company performance to develop the company in the future.\n\nFor long-term investment in manufacturing stocks, investors must consider the exchange rate and the medium to large production index because these variables influence each other significantly in the movement of stock prices. The increase in the production index is an illustration of the improved performance of listed manufacturing companies, this condition is supported by increased output and sales of manufacturing companies, which will increase company profits. Rising corporate profits are a reflection of the company's improved performance. The improvement in performance shows an increase in demand for energy. Fluctuations in exchange rates and increases in the production index are central points in investment considerations.\n\nThe continuous weakening of the rupiah in the short run will determine investment conditions in the future. This factor is due to the dependency on raw materials for production from foreign sources, the amount of which will reduce the value of the rupiah.\n\nSince the end of the cycle of Indonesia's mainstay commodities such as petroleum, the Indonesian economy can be strengthened through the manufacturing sector pathway. To do this, policies to improve infrastructure and transportation need special attention from the government, as improved transportation will strengthen investor confidence to re-invest.\n\nAlthough the manufacturing sector in Indonesia is experiencing liberalization widely and is open to the world of investment, on the other hand, due to the problem of unequal access to electricity it is necessary to consider renewable energy as a companion in fulfilling Indonesia’s energy needs and strengthening the Indonesian economy.\n\n\nData availability\n\nFigshare: Data Panel Manufaktur.csv\n\nhttps://doi.org/10.6084/m9.figshare.13656005.v1 (Rachmawati et al., 2021)\n\nThis project contains the following underlying data:\n\n• Data Panel Manufaktur.csv (Monthly data for January 2009 – December 2016, the operational variable determined in this study is the oil price index which is an index of oil prices (ICP), while ICP oil prices refer to Platts, Rim oil prices.)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Author contributions\n\n\n\nHandri: Designed the research objectives and variables and analyzed the research findings using the tools\n\nHendrati Dwi Mulyaningsih: Determined the research objectives, and analyzed the findings.\n\nAchmad Kemal Hidayat: Interpreted the result and enhance in discussion.\n\nRudi Kurniawan: Interpreted the result and enhance in discussion.\n\nAni Wahyu Rachmawati: Analyzed the findings, prepare manuscript and submission\n\n\nAcknowledgments\n\nThe research team would like to say thank you, to Universitas Islam Bandung Indonesia, Universitas Padjadjaran Bandung Indonesia and Universitas Wanita Internasional Bandung Indonesia.\n\n\nReferences\n\nCai XJ, Tian S, Yuan N, et al.: Interdependence between oil and East Asian stock markets: Evidence from wavelet coherence analysis. J Int Financial Mark Inst Money. 2017; 48: 206–223. Publisher Full Text\n\nChoi I: Unit root tests for panel data. J Int Money Finance. 2001; 20(2): 249–272. Publisher Full Text\n\nDumitrescu EI, Hurlin C: Testing for Granger Non-causality in Heterogeneous Panels To cite this version : HAL Id : halshs-00224434 Testing for Granger Non-causality in Heterogeneous Panels. Econ Model. 2012; 29(4): 450–1460.\n\nFisher RA: Statistical Methods for Research Workers. Oliver and Boyd, Edinburgh. 1932. Publisher Full Text\n\nHamilton JD: Oil and the Macroeconomy. In the New Palgrave Dictionary of Economics. Second edition, ed. Steven N. Durluf and Lawrence E. Blume. Houndmills, U.K. and New York: Palgrave Macmillan. 2008. Publisher Full Text\n\nHurlin C: What would nelson and plosser find had they used panel unit root tests? Appl Econ. 2010; 42(12): 1515–1531. Publisher Full Text\n\nJones CM, Kaul G: Oil and the Stock Markets. J Financ. 1996; 51(2): 463–491. Publisher Full Text\n\nMaddala GS, Wu S: A comparative study of unit root tests with panel data and a new simple test. Oxf Bull Econ Stat. 1999; 61(S1): 631–652. Publisher Full Text\n\nNason GP: Stationary and non-stationary time series. Introductory Econometrics: A Practical Approach. 2013; (1994): 273–285.\n\nPedroni P: Critical values for cointegration tests in heterogeneous panels with multiple regressors. Oxf Bull Econ Stat. 1999; 61(S1): 653–670. Reference Source\n\nRachmawati AW, Handri H, Mulyaningsih HD, et al.: Data Panel Manufaktur.csv. figshare. Dataset. 2021. http://www.doi.org/10.6084/m9.figshare.13656005.v1\n\nRamos SB, Veiga H: Oil price asymmetric effects: Answering the puzzle in international stock markets. Energy Econ. 2013; 38: 136–145. Publisher Full Text\n\nRafailidis P, Katrakilidis C: The relationship between oil prices and stock prices: a nonlinear asymmetric cointegration approach. Appl Financ Econ. 2014; 24: 793–800. Publisher Full Text" }	[ { "id": "93342", "date": "20 Sep 2021", "name": "Kazeem O. Isah", "expertise": [ "Reviewer Expertise Financial Economics & Macroeconomics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper the impact of Indonesian oil price (CPI) and macroeconomics on investments in the manufacturing sector in Indonesia . My comments are:\nFirst, the introduction needs more works to make it an informative introduction. The introduction in its current form lacks substance. I will encourage the author(s) to provide a strong motivation for the study. The way to go is to rewrite the introduction to include research question(s) and then motivate the paper on why it is important to address the research question(s), particularly in the context of the investigated economy.\n\nSecond, the author(s) needs to be specific on their contribution to the literature. In a clear and concise manner, they should identify what is lacking in the literature by way of the limitation of the extant studies and how their study adds to this literature. Saying it differently, what is there in the manuscript being reviewed that can be considered innovative compared to what is available in the previous studies.\n\nThird, the title as well the objective of the paper suggests it is an impact analysis. Hence, I am wondering to what extent is the Granger causality testing procedure is appropriate in the study. Rather a single equation - based namely, panel autoregressive distributed lag model (PARDL) might be more suitable for the objective of the study. More importantly, the author’s preference for a particular model or estimation technique should be strongly motivated in terms of its appropriateness for the possible inherent dynamic and statistical features of the variables under consideration.\n\nFinally, the authors should further add value to their result discussion by relating their findings to those of the earlier studies, how it is different or the same compared to other similar studies done, and what is the implication of the finding?\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "121653", "date": "14 Feb 2022", "name": "Zeravan Asaad", "expertise": [ "Reviewer Expertise The interest of my research: Financial Management", "Financial Economics", "Stock Markets efficiency", "and Foreign Direct Investment", "I am writing to confirm my capability to evaluate the whole aspect of the current paper" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nReviewing Report About the paper entitle (The impact of Indonesian oil price (CPI) and macroeconomics on investments in the manufacturing sector in Indonesia) Below are my comments:\nIntroduction:\nThe introduction is very weak and it looks like general information, hence the introduction should be rewritten to include the following:\nBackground about the study variables such as oil, manufacturing sector, stock market, and the economy of Indonesia.\n\nThe importance of the study and how will benefit from the result for instance.\n\nAdd significance to the study.\n\nAdd problem statement (research gap) to the study.\n\nEach paragraph needs to be supported by references, for example, the first paragraph at least needs six references (Oil is probably the most important source of energy in the economy (Reference), and also an important component of the production cost of companies in the manufacturing sector (Reference). Fluctuations in oil prices will therefore affect the performance of the manufacturing sector (Reference). Disruption of the sector’s performance will also cause fluctuations in the share price of the manufacturing sector (Reference). Furthermore, as far as we know, stock price movements are influenced by internal and external factors caused by macroeconomic conditions (Reference). However, investors can still invest even in unstable macroeconomic conditions but need to be more prudent (Reference).\n\nStructure of study at the end of the introduction.\nLiterature review (Related Work) Missing related work in the study should be supported by many previous studies in the same context and in other contexts,\nAdding a new part of the literature review in the study.\n\nAdd a paragraph showing the differences between the current study with the previous studies.\n\nSupport this part by some studies done in the Indonesian context and studies done in the ASEAN region or emerging or developing countries.\n\nAdding a theoretical connection between the independent variables and dependent variables.\n\nAdding background about the investment sector and stock market in Indonesia.\nMethodology In general methodology part is acceptable but can be better by making more adding the following:\nOperational definition (variable definition)\n\nHypothesis\n\nThe objective of the study\n\nUnderpinning theory of the study.\n\nJustification of choosing the study variables.\n\nThe study mentioned on page 3 (evidence that the variables under study are unit-root variables of order one. In other words, they are integrated of order one), but at the same time mentioned on table 2 that (the test of the panel unit root is stationary at level), so how to solve this contradiction with the studies which mentioned that if non-stationary at level, we need to check cointegration as shown here and panel autoregressive distributed lag model (PARDL) might be more suitable due to panel unit root test.\n\nDiscussion\nIn discussion, the paragraph does not make any comparison between the current study results and previous studies, to identify whether it is consistent or not with the past studies in the literature review in order to support the current study results in the case in the same line with past studies, also to justify the current study results in the case are in contrary with past studies (This study does not have the comparison between the current study results and the previous studies).\nConclusions:\nThe conclusions need to rewrite and add the following:\nAny suggestions for further studies.\n\nAny limitations of the study\nOther:\nCheck the sentence in methods at abstract on page 1 (equilibrium, and short the short run)\n\nCanceling the table (1) on page 4 because it is not necessarily given as they are.\n\nAdd a year to the reference on page 3 (Im, Pesaran, and Shin) and add to the reference list.\n\nUsing academic writing such as this study or current study instead of (our, we …) on pages (3, 4, …..).\n\nDeleting the repeating paragraph on page (5) (Based on panel unit root statistics we proceed to panel cointegration analysis in order to examine long-run relationship among the variables. The first stage is to determine that the relationships among variable are not spurious, the panel unit root result should be tested by the Fisher and Shin method in stationary at level. The result of the panel unit root test can be seen in Table 2 below).\n\nRecheck the whole study Grammarly.\n\nAdding the table of descriptive statistics in the study.\n\nThe researcher said in the study on page 3 in methods (like most of the previous studies…) without indicating or supporting by any references.\n\nMany sentences and paragraphs need to know who said that on pages 3, 4, 5,\n\nRecheck the paragraph (The consumer price index shows an increasing trend until 2012 with an average of 141.66, and after that follows a decreasing trend with an average of 185.2).\n\nThe study did not cover any diagnostics of intertemporal dependencies, autocorrelation, endogeneity, or any other aspects of statistical problems,\n\nThe article should be revised based on the above comments before indexing.\n\nWish all the best to the researcher.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-338
https://f1000research.com/articles/10-35/v1	19 Jan 21	{ "type": "Research Article", "title": "Cytotoxic T cells response with decreased CD4/CD8 ratio during mammary tumors inhibition in rats induced by non-contact electric fields", "authors": [ "Firman Alamsyah", "Rarastoeti Pratiwi", "Nisrina Firdausi", "Jessica Irene Mesak Pello", "Subekti Evi Dwi Nugraheni", "Ahmad Ghitha Fadhlurrahman", "Luthfi Nurhidayat", "Warsito Purwo Taruno", "Rarastoeti Pratiwi", "Nisrina Firdausi", "Jessica Irene Mesak Pello", "Subekti Evi Dwi Nugraheni", "Ahmad Ghitha Fadhlurrahman", "Luthfi Nurhidayat", "Warsito Purwo Taruno" ], "abstract": "Background: Breast cancer is the most common cancer in women worldwide and is the leading cause of death in women with cancer. One novel therapy used for breast cancer treatment is non-contact electric fields called electro-capacitive cancer therapy (ECCT) with intermediate frequency (100 kHz) and low intensity (18 Vpp). The objective of this study was to examine the effect of ECCT on mammary tumors growth in rats and observing the immune responses that play a role in fighting the tumor. Methods: Female SD rats were used and divided into four groups, namely control (NINT), placebo (NIT), non- therapy (INT), and therapy (IT) groups with 6 biological replicates in each group. Rats in INT and IT groups were treated with 7,12-dimethylbenz[a]anthracene for mammary tumor induction. Only rats in NIT and IT groups were exposed to ECCT individually for 10 hours per day for 21 days. The size of all tumors was measured with a digital caliper. The distributions of PCNA, ErbB2, caspase-3, CD68, CD4 and CD8-positive cells were observed with immunohistochemistry and scoring with ImageJ. Results: The growth rate of mammary tumors in IT group was significantly lower (p<0.05) than that in the INT group. The number of mitotic figures and the percentage of PCNA, caspase-3, and CD68- positive cells in IT group were significantly lower (p<0.05) than those in INT group. Conversely, the percentage of CD8-positive T cells in IT group was significantly higher (p<0.05) than that in INT group. Moreover, the CD4/CD8 ratio in IT group was decreased. Some tumor tissues were blackened and detached from the surrounding tissue, resulting in an open wound which then healed up upon exposure. Conclusions: Non-contact electric fields exposure showed inhibition on mammary tumor growth in rats while inducing CD8+ T cells that lead to tumor cells death and potentially helps wound healing.", "keywords": [ "non-contact electric fields", "ECCT", "mammary tumors", "CD8+ T cells", "CD4/CD8 ratio" ], "content": "Introduction\n\nBreast cancer is the most common cancer among women in the world, in which 1 in 8 women in the world is at risk of developing breast cancer1. Furthermore, about 2.1 million new cancer cases in 2018 are breast cancer cases and this type of cancer is the leading cause of death in women with cancer2. Commonly, the treatment of breast cancer is carried out in two ways. The first, termed local therapy, controls or removes the tumor in specific areas, such as through breast-conserving therapy and mastectomy that may be followed by radiotherapy. The second, called systemic therapy, uses the circulatory system in the body to inhibit cancer cells growth that spread in parts of the body. This type of treatment includes chemotherapy, targeted therapy, and endocrine therapy3. However, breast cancer therapy applied so far often causes negative effects, such as the presence of pathophysiological mechanisms that cause chronic pain4, or the mechanism of multidrug resistance (MDR) in breast cancer cells as a reaction to chemotherapy5,6. Moreover, breast cancer therapies may attack certain normal cells or tissues that result in toxicity symptoms7. A novel cancer therapy based on electric field exposure with intermediate frequency and low intensity called Tumor Treating Fields (TTFields) has been developed to prevent such negative effects of cancer therapy and still inhibit cancer growth. No toxic side effects or adverse events were found on animal tumor models nor patients under the exposure to this electric field-based cancer therapy, except for skin toxicity or dermatitis of low severity8,9.\n\nSince skin toxicity or dermatitis arises in electric field therapy due to prolonged direct contact between the electrodes and the skin10, we have developed a type of non-contact electric field therapy using capacitive modality to avoid such injuries. In our previous study in preclinical setting, cancer therapy namely Electro-Capacitive Cancer Therapy (ECCT) using the non-contact electric fields with intermediate frequency (100 kHz) and low intensity (18 peak-to-peak Voltage/Vpp) could reduce the size of breast tumors in mice significantly without causing histological damage to mammary and skin tissues. In addition, lymphocyte and macrophage infiltration was found around the tumor area through the blood vessels11. One of the mechanisms of tumor cells death can be carried out with the act of anti-tumor mechanism of various immune cells, including helper and cytotoxic T cells of lymphocytes, as well as macrophages12. Cytotoxic CD8+ T cells which is activated by helper CD4+ T cells, may enter the tumor microenvironment and induce apoptosis, resulting in shrinkage of the tumor lesions12,13. Macrophages with the M1 phenotype play an important role in the recognition and destruction of cancer cells and their presence usually indicates a favorable prognosis. Whereas macrophages with the M2 phenotype are involved in anti-inflammation, helping the angiogenesis process and the expression of scavenger receptors, and have a significant role in tumor development and metastasis12,13, including breast tumor14,15. One of the marker proteins to observe the infiltration of macrophages in a tissue, including mammary tumor tissue, is the CD68 protein14–16.\n\nCancer therapy that can re-induce immune response to destroy and clean up tumor cells is a therapy that is being developed because of its positive effects. By activating immune cells, the immune cells may reach the tumor-specific areas, including metastatic area, and specifically attacking only tumor cells, not the normal cells in the vicinity17. Petri et al.18 reported that static electric fields increase the immune response in mice. Moreover, electric fields modulate the activation and polarisation of some immune cells, including T cells19 and macrophages20. Voloshin et al.21 reported that TTFields exposure combined with anti-PD-1 therapy induced immunogenic cell death in lung and colon tumors. In addition, Pratiwi et al.22 reported the increased expression of CD68 and caspase-3 in rat breast tumor tissues under the exposure to 150 kHz non-contact electric fields while reducing the expression of PCNA and ErbB2 proteins. However, the study by Pratiwi et al.22 showed an increase of the tumor size during therapy. Using different intermediate frequency (100 kHz), we examined the effect of non-contact electric fields exposure on tumor growth in mammary tumors-induced rats and observed the activation of immune cells in fighting the tumor cells. We hypothesized that the increase in the size of mammary tumors would not happen under exposure to this non-contact electric fields with the induction of immune cells, especially lymphocytes and macrophages.\n\n\nMethods\n\nThis study was conducted at the Integrated Research and Testing Laboratory (LPPT) of Universitas Gadjah Mada (UGM) and at the Animal Structure and Development Laboratory of Faculty of Biology, UGM. LPPT UGM has been awarded ISO/IEC 17025:2000 accreditation for the competence of testing and calibration22. Experimental protocol in this study was performed following approval by the Ethical Clearance Committee of LPPT UGM with ethical clearance number: 00015/4/LPPT/IV/2017. The Ethical Clearance Committee stated that this study has been declared to meet the ethical requirements for research on experimental animals and the Ethical Clearance Committee has the right to conduct monitoring during the research.\n\nA total of 40 5-week-old healthy female Sprague Dawley (SD) rats (Rattus norvegicus, Berkenhout 1769) weighing 50−80 gram were used for this study. SD rats have been used as animal tumor model to study human breast cancer with various induction agents, as well as with DMBA administration23,24, since rats have 98% genetic homology with humans25. The rats were supplied by LPPT UGM laboratory, Yogyakarta, Indonesia, and never used for any other experiment. Rats showing any symptoms of illness or abnormalities were excluded from the experiment. The rats were acclimated for 1 week in polypropylene cages as communal home cages with each size: 50 × 40 cm2 that covered with rice hulls bedding at the bottom. One communal cage consisted of 5 rats. The temperature of the animal room was maintained at between 23–26°C with 81.09% average relative humidity to avoid dehydration during electric fields exposure, lighting condition was light from lamps during the day and total darkness during the night (12L:12D photoperiod). The rats were fed with AD2 pellet animal diet and tap water ad libitum. However, during treatment, cucumber slices were given as a substitute for water, to avoid the animals being shocked with electricity, since the tip of the drink bottle contains metal which can permit electric fields. Cage cleaning was carried out every day by cleaning the animal waste from the cage and changing food and water. Individual marking was conducted by giving picric acid to rat fur, while group marking was conducted by labelling the cage with a paint marker to avoid potential confounders.\n\nThe SD rats were divided into four groups, namely control (non-induction and non-therapy/NINT), placebo (non-induction and therapy/NIT), DMBA-induced mammary tumors without therapy (induction and non-therapy/INT), and DMBA-induced mammary tumors with therapy (induction and therapy/IT) groups. The sample size in each group was calculated according to the Federer formula, where 6 biological replicates were used for each group22. The animals were selected randomly and assigned to control and treatment groups. Rats were picked randomly for mammary tumors induction with a single dose of DMBA (20 mg/kg body weight) administration two times per week for 5 weeks. The first 6 rats to develop tumors were assigned to IT group, and then 6 more rats were assigned to INT group. Not all rats grew tumors after DMBA administration. All healthy, normal rats in the placebo (NIT) group were given corn oil as DMBA (7,12-dimethylbenz[a]anthracene) solvent. DMBA compound is a neuroendocrine disruptor that commonly used for carcinogenesis in specific organs, including breast tumors in rats26,27. All administrations were performed by the same researcher (AGF). Each researcher in the team had different task. Besides the researcher who administered DMBA, other researchers measured the size of the nodules, recorded the morphology of mammary tumors, dissected the euthanized rats and analysed the data statistically. One researcher (FA) controlled and monitored all the different steps in the experiment.\n\nFollowing the development of mammary tumors up to 1.5 cm in length, all rats in the IT group were exposed to intermediate frequency (100 kHz) and low intensity (18 Vpp) non-contact electric fields generated between pairs of capacitive electrodes embedded in individual cages that had been modified to an ECCT device (Figure 1a). The device is called non-contact because its electrodes do not directly attach to the animal skin (Figure 1b). All individual cages were placed on the same shelf at the same height. Non-contact electric fields therapy was performed 10 hours per day for 21 days with 2 hours rest after first 5 hours therapy with the same starting time of therapy each day. The therapy was stopped when the mammary tumors has increased to 2.25 cm2 in size or it has reached the last day of therapy. All rats in placebo (NIT) group were also exposed to non-contact electric fields in the same period, 3 weeks after the last corn oil administration. The duration of electric fields exposure for 10 hours per day was shorter than the previous exposure in our preliminary study11 to minimize any possible side effects due to exposure to electric fields8,10. After treatment, all animals were returned to their communal cages.\n\n(a) ECCT with oscillators connected to electricity, (b) A rat inside an individual cage of ECCT. The dimension of the cage was 23 cm × 18 cm × 19 cm. The arrangement of the electrodes has been described in our previous study11.\n\nDuring therapy, all tumors were palpated once every 2 days and their size (cm2) was measured with a digital caliper and tabulated. All nodule measurements were conducted by the same researcher (NF). In this study, we did not measure nodule size in volume due to tool limitations. The tumor size data were used to calculate the tumor surface area (TSA) using the formula for circle and ellipse areas, and subsequently the TSA data were used to calculate the tumor growth rates (TGR) of mammary tumors. The TGR was calculated by using specific growth rates (SGR) formula28 with modification as follows: SGR (%/day) =ln (A2/A1)/(t2 – t1), where A1 and A2 are the tumor surface area before (t1) and after therapy (t2), respectively. Besides tumor size, the morphology of mammary tumors was also recorded by the same researcher (JIMP).\n\nAfter therapy was completed, all rats were euthanized by an overdose of ketamine (150 mg/kg of body weight) for deep anesthesia using intramuscular injection. The rats were pinned ventral side up on a dissected box and dissected with the same surgeon (AGF). Nodules and normal breast tissues were taken and washed using physiological saline. All tissues were fixed using neutral buffered formalin (NBF). Three nodules from three different rats were collected from each INT and IT groups, while one normal breast tissue was collected from each NINT and NIT groups. These six nodules were used for histological examination with immunohistochemical staining (IHC) of the target antigens. While the normal breast tissues were used for comparison of the overall histological structure without IHC.\n\nHistological examination was performed by using paraffin-embedded Hematoxylin and Eosin (H&E)-stained histology slides followed by IHC29 using Starr Trek Universal HRP Detection Kit Biocare. Observations and counting were performed on the mitotic figures on H&E preparations. Six antibodies purchased from Abcam Cambridge, United Kingdom, were used for IHC, namely anti-PCNA polyclonal antibody (Cat# ab18197, RRID:AB_444313), anti-ErbB2 monoclonal antibody (Cat# ab16901), anti-caspase-3 polyclonal antibody (Cat# ab13847), anti-CD68 monoclonal antibody (Cat# ab201340), anti-CD4 polyclonal antibody (Cat# ab203034), and anti-CD8 alpha monoclonal antibody (Cat# ab33786, RRID:AB_726709). The dilution for anti-CD4 and anti-CD8 antibodies was 1:750, where 1 µl of antibody was diluted with 750 µl of BSA-PBST solution (Bovine Serum Albumin 1% - Phosphate Buffered Saline with Tween® 20). While the dilution for anti-PCNA, anti-ErbB2, anti-caspase-3 and anti-CD68 antibodies was 1:500. As much as 100 µl diluted primary antibody was applied on the appropriate slide covered with a parafilm and placed in a humidity chamber. Then, the slide was removed to a refrigerator (4°C) overnight. One to two drops (50 to 100 µl) of biotinylated secondary antibody Trekkie Universal (Biocare Medical, STUHRP700 H L10) were applied on the slides covered with a parafilm and incubated for 30 minutes at room temperature. The concentration of antibodies given was followed the manufacturer’s instructions.\n\nAll measured data were analysed with appropriate methods without any exclusion. Semi-quantitative IHC scoring was performed using the percentage of positive area of stained cells (CD4 and CD8-positive T cells; caspase-3, ErbB2, PCNA-positive tumor cells) and the number of stained cells (CD68-positive macrophages). IHC scoring was performed in ImageJ ver.1.51, a freely available software22. The comparison between positive area of stained cells and total area showed the distributions of targeted antigens and cells30. Scoring was carried out in 50 fields per group (INT and IT groups), in which 17–18 random fields were examined in one slide. In addition, CD68+ macrophages counting was performed by using 60 fields per group with 20 random fields examined in a slide. In total, three slides from three different nodules were examined from each group.\n\nAll data were tested for normality (Shapiro-Wilk test, α = 0.05). The data of mammary tumors growth rate from 12 animals (6 rats each from IT and INT groups) and the number of mitotic figures were analysed using T-test (α = 0.05) to find statistical significance. The data of PCNA, ErbB2, caspase-3, CD68, CD4, and CD8-positive cells and CD4/CD8 ratio were analysed quantitatively using non-parametric Mann-Whitney test, since the data were not normally distributed. All data were analysed statistically using GraphPad Prism ver.8.4.3 software22 for Windows by the same researcher (NF). Qualitative data (morphology of mammary tumors) were analysed descriptively.\n\n\nResults\n\nThe outcome of this study was the growth rate of mammary tumors under exposure to non-contact electric fields. Comparison of histological characteristics between breast tissue and breast cancer in the animal tumor model, the distribution of molecular markers that described the proliferation and the mechanism of cell death, and the response of immune cells under exposure, as will be explained coherently below.\n\nThe growth rate of rat mammary tumors in IT group (-0,043 ± 0,065) was significantly lower (p<0.05) than that in INT group (0,108 ± 0,078) and showing negative tumor growth rate as shown in Figure 2a. There was no tumor growth in the placebo (NIT) group. Mammary tumors morphology in INT and IT groups showed differences, in which mammary tumors in the INT group were generally more compact as previously reported by Pratiwi et al.22 and appear bruised (Figure 2b) due to thrombocytopenia from the malignancy31, whereas mammary tumors in the IT group were generally softer because they contained fluid22 and some were blackened (Figure 2c). The blackened mammary tumors in the IT group were released from the surrounding tissue, as shown in Figure 3a. These tumors initially darkened, then hardened like a scab as occurs in a dry wound, and slowly sloughed off from the surrounding tissue, causing an open wound which then dried up (Figure 3b) and shrank upon exposure to non-contact electric field (Figure 3c).\n\n(a) SGR of mammary tumors in INT and IT groups, (b) morphology of a mammary tumor in INT group with the appearance of bruises, (c) morphology of a mammary tumor in IT group with a blackened condition. Different letters (a, b) on top of the charts indicate significant differences (p<0.05).\n\n(a) open wound on day 14 of therapy, (b) open wound on day 21 (final day) of therapy, (c) the reduction of wound size from day 14 to day 21 of therapy.\n\nThe characteristics of normal breast tissue and the pathogenesis of actively dividing cells in DMBA-induced breast cancer in rats can be seen through H&E staining (Figure 4). Figures 4a, b show microscopical sections of breast tissue from rats of NINT and NIT groups, respectively, having normal histological features with the presence of adipose tissue and other connective tissues, and in the absence of actively proliferating cells. In addition, a myoepithelial cell layer was clearly visible in the glandular epithelium32. Conversely, Figure 4c, d show the level of malignancy of breast cancer cells from rats of INT and IT groups, respectively. They show histological feature of papillary carcinoma with the characteristics of malignant epithelial proliferation protruding into the lumen of the glandular duct that made it constricted. However, breast cancer in rats of the IT group tend to be less malignant than that in the INT group, indicated by the clear glandular duct and glandular lobule lumens, and the prominent epithelial structure as well (Figure 4d). In addition, the lumens in IT group still maintained an intact layer of cells (Figure 4d), while most of the lumens in INT group did not (Figure 4c), due to the pressure exerted by the breast cancer cells. Moreover, the region of adipose tissue and other connective tissues were also pressed by the mammary tumor cells (Figure 4d). On the other hand, the tumor tissue itself undergoing necrosis (Figure 4c, d) which was characterized by inflammation33,34 and the necrotic areas with inflammatory environment in the INT group was larger than that in the IT group. Figure 4e and Figure 4f show mitotic figures and apoptotic bodies in INT and IT groups, respectively.\n\n(a) Control (NINT) group, (b) Placebo (NIT) group, (c) Non-therapy (INT) group, (d) Therapy (IT) group, (e) Mitotic figures (indicated by arrows) and apoptotic bodies (in circles) in INT group, and (f) Mitotic figures and apoptotic bodies in IT group. CT= Connective tissue, NA= Necrotic area, DEC= Ductal epithelial cells, MEC= Myoepithelial cells, LU=Lumen, AC= Adipose Cells, BV = Blood Vessel.\n\nImmunohistochemical staining with anti-PCNA, anti-ErbB2 and anti-caspase-3 detected PCNA, ErbB2, and caspase-3-positive cells, respectively, in rat mammary tumors. The expression of PCNA in INT and IT groups were detected in the nucleus of tumor cells (Figure 5a and Figure 5b, respectively), while the expression of ErbB2 in both groups was found on the cell membranes (Figure 5c and Figure 5d, respectively). Caspase-3 was expressed in different locations in mammary tumors of INT and IT groups (Figure 5e and Figure 5f, respectively). The expression of caspase-3 in INT group was found in the cell cytoplasm (Figure 5e), while in IT group, caspase-3 expression was found in the nucleus (Figure 5f) and resulted in chromatin condensation and DNA fragmentation, indicating that apoptosis has reached a late stage35,36. In addition, the expression of caspase-3 in IT group was also found at hollow regions in mammary tumors (Figure 5f), whereas caspase-3 expression in INT group was rarely found in the same region (Figure 5e).\n\n(a) Expression of PCNA in the nucleus of tumor cells in non-therapy (INT) group, (b) Expression of PCNA in the nucleus of tumor cells in therapy (IT) group, (c) Expression of ErbB2 on the cell membranes of tumor cells in INT group, (d) Expression of ErbB2 on the cell membranes of tumor cells in IT group, (e) Expression of caspase-3 in the cytoplasm of tumor cells in INT group, (f) Expression of caspase-3 in the nucleus and Hollow regions (HR) of tumor cells in IT group. Brown color chromogen and arrows indicates positively stained cells.\n\nBreast cancer cells that actively proliferated in rats of the INT group were seen to be relatively greater than those in the IT group, indicated by the higher number of mitotic figures per field in the INT group (10.14±3.80) compared to that in the IT group (5.43±1.81) as shown in Figure 6a. In addition, the percentage of positively stained areas of PCNA in INT group (36.16 ± 5.16%) was significantly higher than that in INT group (23.65 ± 3.84%, Figure 6b). Figure 6c and Figure 6d show that INT group had a greater percentage of positively stained areas of ErbB2 (0.045 ± 0.029%) and caspase-3 (8.90 ± 5.48%) than those in IT group (0.036 ± 0.029 for ErbB2 and 3.01 1.52% for caspase-3). However, the difference of percentage of positively stained areas of caspase-3 between the two groups was considered statistically significant (p<0.05), while the difference in ErbB2 expression was not significant (p>0.05).\n\n(a) Number of mitotic figures, (b) percentage of PCNA-positive cells, (c) percentage of ErbB2-positive cells, (d) percentage of caspase-3-positive cells. Error bars represent the standard error of the mean. Different letters (a, b) on top of the charts indicate significant differences (p<0.05).\n\nImmunohistochemical staining with anti-CD68, anti-CD4 and anti-CD8 detected CD68+ macrophages, CD4+ and CD8+ T cells, respectively, in rat mammary tumors. The expression of CD68, CD4 and CD8 were detected on the cell membrane and in cytoplasm (Figure 7). In addition, CD68+ macrophage infiltration was found in mammary tumors of INT and IT groups, particularly in necrotic areas and hollow regions (Figure 7a and Figure 7b, respectively). The hollow regions in IT group were seen to be more numerous and larger in size than those found in INT group. In addition, CD68+ macrophage infiltration into the hollow regions of mammary tumors in IT group were also seen to be greater than that in INT group. Conversely, CD68+ macrophages infiltration in necrotic areas was seen to be higher in INT group (Figure 7a) than that in IT group (Figure 7b), since the distribution of necrotic areas in INT group was also wider than that in IT group. Similarly, CD4+ and CD8+ T cells were also found around the proliferating tumor cells that may respond to certain antigens in the mammary tumor cells, within blood vessels, and in the necrotic areas12,13 which were characterized by inflammation33,34 (Figure 7c and Figure 7d for CD8, Figure 7e and Figure 7f for CD4).\n\n(a) CD68+ macrophages in non-therapy (INT) group, (b) CD68+ macrophages in therapy (IT) group, (c) CD8+ T cells in INT group, (d) CD8+ T cells in IT group, (e) CD4+ T cells in INT group, (f) CD4+ T cells in IT group, Brown color chromogen indicates positively stained cells. MC: Macrophage cells, CT: Connective Tissue, BV: Blood Vessel, DEC: ductal epithelial cells, NA: Necrotic area, CTL: Cytotoxic T Lymphocyte, and HTL: Helper T Lymphocyte.\n\nSignificantly higher number (p<0.05) of CD68+ macrophages was found in the INT group (8.20 ± 0.52) than that in IT group (4.87± 0.30, Figure 8a) with lower growth rate (Figure 2a). The percentage of positively stained areas of CD4 in INT group (0.42 ± 0.08%) was higher than that in IT group (0.32 ± 0.08%), but the difference was not statistically significant (p>0.05, Figure 8b). In contrast, the percentage of positively stained areas of CD8 in INT group (0.21 ± 0.04%) was significantly lower (p<0.05) than those in IT group (0.64 ± 0.11%, Figure 8c). Consequently, the ratio of CD4/CD8 in INT group (7.16 ± 2.60) was higher than that in IT group (2.44 ± 1.08, Figure 8d).\n\n(a) Number of CD68-positive macrophages, (b) percentage of CD4-positive T cells, (c) percentage of CD8-positive T cells, (d) ratio of CD4/CD8. Error bars represent the standard error of the mean. Different letters (a, b) on top of the charts indicate significant differences (p<0.05).\n\n\nDiscussion\n\nIn the present study, the significantly lower growth rate of rat mammary tumors in the IT group with a negative mean value of SGR compared to INT group with a positive mean value of SGR (Figure 2a) showed the inhibitory effect of non-contact electric fields of ECCT against mammary tumor cells that are actively dividing in the animals, as we have previously reported in our preliminary study in mice11. In addition, the development of mammary tumors exposed to 100 kHz non-contact electricfields of ECCT was decreased, whereas in the study of Pratiwi et al.22 which used a frequency of 150 kHz, it was increased. This may suggest that differences in the frequency of the electric fields can give different therapeutic results in cancer37,38 and the 100 kHz was the appropriate frequency of non-contact electric fields in inhibiting the proliferation of mammary tumor cells.\n\nThe absence of tumors in the placebo (NIT) group (Figure 4b) demonstrated the safety of non-contact electric fields of ECCT to healthy or normal animal models, as we have previously reported11. In addition to its safety, this electric fields exposure did not produce acute damage to rat skin tissue or chronic pain which usually occurs after radiation therapy, including breast cancer radiation therapy4,39,40. Instead, it helped wound healing after cancer treatment in rats of IT group, indicated by the drying wound and the reduction of wound size (Figure 3b and Figure 3c). Endogenous electric fields are known to play as a signal that direct cell migration in epithelial wound healing and external electric fields exposure may accelerate wound healing by directing the migration of T cells19 and fibroblast, as well as their proliferation and transdifferentiation41,42. In addition, Hoare et al.20 reported that external electric fields may contribute to the coordination and regulation of macrophage functions, including wound healing. The effect of non-contact electric fields of ECCT in directing immune cells was also seen in the fluid contained in the mammary tumors of the IT group as indicated by the presence of macrophages and lymphocytes infiltration into the tumor area (Figure 7) as we have previously reported11,22. This immune cell infiltration can promote the destruction of cancer cells12,21,43 as demonstrated in the IT group (Figure 2a and Figure 2c).\n\nThe cellular effects of electric fields exposure on PCNA expression during S-phase may indicate the incidence and the growth rate of mammary tumors44. Since PCNA is a S-phase marker in interphase that has a central role in DNA replication and repair45, the decrease of PCNA expression in mammary tumor cells in IT group (Figure 6b) may indicate a decrease in the incidence and the growth rate of mammary tumors44. In addition, alternating electric fields exposure with intermediate frequency and low intensity used in this study can cause tumor cells that are dividing to experience uneven segregation of chromosomes and mitotic catastrophe which followed by programmed cell death46. This is supported by the study of Mujib et al.47 which reported that p53 protein can serve as a marker for cancer cell death via apoptosis due to non-contact electric fields exposure. Moreover, the hollow cavities, also called dead cell islands, with cells debris, may be the spaces that used to be the place for mammary tumor cells that then died through apoptosis48,49, since caspase-3 was found in this place in the IT group (Figure 5f). In addition, within these dead cell islands, the expression level of caspase-3 was very low49, as we found in the IT group (Figure 6d).\n\nThe lower expression of caspase-3 in the IT group (Figure 6d) may suggest the mechanism of mammary tumor cell death under exposure to non-contact electric fields of ECCT through caspase-independent cell death. Meggyeshaz et al.50 reported tumor destruction by electric fields exposure and concomitant heat with elevated DNA fragmentation and low level of caspase-3 expression localized to inflammatory cells which dominantly follows a caspase-independent pathway. Meanwhile, higher caspase-3 expression in INT group (Figure 6d) suggested that higher apoptosis events may have occurred in this group, resulted from oxidative stress due to elevated reactive oxygen species in mammary tumors51,52. Although apoptosis occurred in the mammary tumors, the tumor cell death rate was relatively much lower than the proliferation rate, so that mammary tumor size increased53, as seen in the INT group (Figure 2a and Figure 2b).\n\nThe type of receptor possessed by breast cancer cells in this study was the ErbB2 receptor (Figure 5c and Figure 5d). The generation of mammary tumors with ErbB2 receptors were the results of carcinogenesis due to DMBA administration into the animal tumor model54. Despite such insignificant results in the expression level of ErbB2 between INT and IT groups (Figure 6c), tumor size in the INT group continued to increase from baseline, and its tumor growth rate was higher than that in the IT group (Figure 2a). In addition, the number of mitotic figures and the percentage of PCNA-positive tumor cells in the INT group were also higher than those in the IT group (Figure 6a and Figure 6b, respectively). This suggests that INT group experienced higher tumor cell proliferation than IT group. Moreover, the higher number of CD68+ macrophages as one of inflammatory cells seen in the tumor microenvironment of INT group (Figure 7a and Figure 8a) was capable to induce tumor growth by contributing to cellular migration of tumor cells and metastasis12,14,55. Ong et al.56 also reported that rat mammary carcinoma had significantly higher percentage of CD68+ macrophages compared to benign mammary tumors with a lower growth rate. CD68+ macrophages that infiltrate to the necrotic areas of INT group (Figure 7a) may be considered as M2 macrophages which play a role in tumor growth and provide a poor prognosis12,56,57. In addition, increased M2 macrophages or Tumor-associated macrophages (TAMs) infiltration in mammary tumors would be accompanied by an increase in tumor size14,15,58, as shown in the INT group (Figure 2a and Figure 8a). Conversely, the lower the number of infiltrating TAMs in rat mammary tumors, the smaller the size of the tumors12,15, as shown in the IT group (Figure 2a and Figure 8a). Moreover, the expressions of chemokine (C-C motif) ligand 2 (CCL2) and interleukin 18 (IL18) that have main roles in the development of metastatic breast cancer and excessive angiogenesis by amplifying M2 macrophage polarization59,60 were down-regulated under the exposure to non-contact electric fields of ECCT at 150 kHz frequency22. Similarly, non-contact electric field (100 kHz) exposure in our study may also inhibit the amplification of M2 macrophage polarization resulted in the negative mammary tumor growth rate in rats of IT group (Figure 2a). The infiltration of CD68+ macrophages in mammary tumors of IT group suggested that exposure to non-contact electric fields of ECCT may direct macrophages, likely of M1 phenotype, to the tumor areas11,21 and then engulf and digest dead cell, debris and tumor cells12,61 that eventually produced hollow cavities48,49 as seen in Figure 7b. This macrophage-mediated clearance of dead cells was occurred in caspase-3-independent death62, since the IT group had lower expression of caspase-3 than the INT group (Figure 6d).\n\nImmunogenic tumor cell death induced by the electric fields exposure in the IT group can also be caused by the interaction of lymphocyte with receptors on membrane of mammary tumor cells with the production of cytokines21,46. The distribution of CD4+ and CD8+ T cells within blood vessels and mammary tumors of IT group revealed that non-contact electric fields of ECCT may direct the spreading of these lymphocytes from blood vessels to tumor areas11,19,21, including necrotic areas in it (Figure 7d and Figure 7f). The presence of CD4+ T cells in mammary tumors would induce the activation of CD8+ T cells to kill the tumors12,63,64 as an adaptive immune response12,13,65. However, since the percentage of CD4+ T cells in INT and IT groups was not significantly different (Figure 8b), the presence of CD4+ T cells in tumor areas may not always indicate tumor elimination, instead they may indicate tumor progression12,30,66. Moreover, electric field exposure can decrease CD4+ T cells polarisation19, resulted in the lower percentage of CD4+ T cells in IT group compared to INT group (Figure 8b). Conversely, the presence of CD8+ T cells in the necrotic areas which characterized by inflammation33,34, indicated their role in the inflammatory response to clear dead cells12,67 recognized as foreign agents13. In addition, a higher percentage of CD8+ T cells in IT group (Figure 8c) indicated increased anti-tumor immunity and immunological ability against tumor progression66,68,69. CD8+ T cells may release granzymes during granule exocytosis when tumor cells are marked for elimination12,13,70, and the granzymes may induce caspase-independent apoptotic pathways71,72 which support the presence of hollow cavities in the tumor areas with low caspase-3 expression49 as seen in the IT group (Figure 5f and Figure 6d).\n\nThe ratio of CD4/CD8 is correlated to clinical aspects and could be used as prognostic factor for breast cancer, where higher CD4/CD8 ratio in INT group (Figure 8d) was correlated to mammary tumor progression73. Conversely, the higher positive area of CD8+ T cells and the lower ratio of CD4/CD8 in IT group suggested a good prognosis in rat breast cancer12,66,73 with non-contact electric field exposure. Further study will be conducted to investigate the potency of non-contact electric fields exposure in wound healing upon breast cancer treatment by observing the presence of cytokines, as well as the migration of immune cells and fibroblast that regulate wound healing20,41,42 by using tissue samples from the IT group. Based on the evidence of its efficacy and safety on preclinical studies, the 100 kHz non-contact electric field has been used in phase I clinical trials of ECCT for healthy volunteers.\n\n\nConclusions\n\nIn summary, low intensity (18 Vpp) and intermediate frequency (100 kHz) non-contact electric fields exposure showed inhibition of mammary tumor growth in rats by inducing CD8+ T cells that lead to tumor cells death supported by decreased CD4/CD8 ratio. It also showed inhibition of M2 macrophage polarization resulted in the negative tumor growth rate. The proposed therapy gives good prognosis in rats with mammary tumors and potentially helps in wound healing of the tumor aftermath during the treatment.\n\n\nData availability\n\nOpen Science Framework: Cytotoxic T cells response with decreased CD4/CD8 ratio during mammary tumors inhibition in rats induced by non-contact electric fields, https://doi.org/10.17605/OSF.IO/3P79474 (registered on 11th December 2020: https://osf.io/b6d4m).\n\nThis project contains the following underlying data:\n\n- Ethical Clearance document\n\n- Growth rate of mammary tumors and wound healing data\n\n- IHC scoring data\n\n- Raw images for IHC and hematoxylin figures\n\n- Statistical analysis with GraphPad Prism ver.8.4.3\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nWe would like to thank Mrs. Ardaning Nurliani from Faculty of Biology of Universitas Gadjah Mada for helping the antibodies procurement.\n\n\nReferences\n\nSiegel R, Naishadham D, Jemal A: Cancer Statistics, 2012. 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Reference Source\n\nAlamsyah F: Cytotoxic T cells response with decreased CD4/CD8 ratio during mammary tumors inhibition in rats induced by non-contact electric fields. 2020. http://www.doi.org/10.17605/OSF.IO/3P794" }	[ { "id": "79321", "date": "23 Feb 2021", "name": "Bilgin Keserci", "expertise": [ "Reviewer Expertise Medical imaging", "cancer therapy", "MRI", "image guided therapies" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nEven though the study results are quite impressive, I have some additional issues to be considered/corrected before this paper is indexed.\nFirst, the manuscript needs serious language editing. I strongly advise them to get a professional editing service when revising.\n\nSecond, the below concerns must be answered and explained well in the manuscript before being indexed:\nOn page 4 it says, \"The first 6 rats to develop tumors were assigned to IT group, and then 6 more rats were assigned to INT group. Not all rats grew tumors after DMBA administration. All healthy, normal rats in the placebo (NIT) group were given corn oil as DMBA (7,12-dimethylbenz[a]anthracene) solvent.\"\nQuestions:\nWhat does this mean? How many rats developed tumors in the IT and INT groups? Why were all the rats that developed the tumor first included in the IT group rather than equally distributed to INT and IT?\n\nWere the rats in the NIT group taken from the non tumor growing induced group after being given corn oil?\nOn page 5 it says, \"Three nodules from three different rats were collected from each INT and IT groups, while one normal breast tissue was collected from each NINT and NIT groups.\"\nQuestion:\nWhy not all? How are the rats chosen?\nOn page 7 it says, \"In addition, the percentage of positively stained areas of PCNA in INT group (36.16 ± 5.16%) was significantly higher than that in INT group (23.65 ± 3.84%, Figure 6b).\"\nComment:\n'INT' in bold above is supposed to be 'IT' group. Please correct it.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6398", "date": "01 Mar 2021", "name": "Firman Alamsyah", "role": "Author Response", "response": "1. We will consider getting a professional language editing service to revise our manuscript. 2. The rats were randomly selected for mammary tumor induction with DMBA administration. One month after DMBA administration, the tumors began to grow with a different time of appearance which was between 38-63 days. Since we used 6 biological replicates for each group based on Federer formula, 12 rats were used for IT and INT groups. Then, we selected the first 6 rats that developed tumors to be assigned to the IT group, so that it was technically easier to carry out the research and avoided mistakes in operating the ECCT device. We did not equally distribute the induced rats to both groups to avoid human errors in operating the ECCT, since we had to turn on and off the device twice every day for the IT group, while not for the INT group. 3. The rats in the NIT group were normal rats with corn oil administration. They did not experience any DMBA administration. 4. We used 3 nodules from 3 rats for each IT and INT group, since this sample size was sufficiently representative for the IHC method. We randomly selected the rats with nodules for histopathological analysis. 5. Yes, we made a mistake for that sentence. We will correct it." } ] }, { "id": "79324", "date": "03 Mar 2021", "name": "Elfahmi Elfahmi", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article consists a quite good scientific background and methodology and has an innovation to be contributed in the treatment of cancer, however other clinical studies should be conducted. The quality of works and article is suitable for indexing, however, some comments should be addressed by author for the statement or question as follow:\nSome data presented in the results showed a quite big deviation, are there any outliers in the 6 replicates data which contributed for this deviation? If it’s possible that the authors could explain this clearly, it would then strengthen the paper.\n\nAlthough the electrical field is not contacted with the animal models, it is placed in the closed chamber. Could the authors explain in scientific view whether there are no side effects of the electric field?\n\nThe condition parameter of electric field used in this study is only point. Is it an optimum condition? If yes, the proven study for the optimalization should be included.\n\nIf there is toxicity data of the instrument published, it would be better if included.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6519", "date": "04 May 2021", "name": "Firman Alamsyah", "role": "Author Response", "response": "1. The deviation value that is quite large in some data shows a quite different response from each individual rat to the electric field exposure. For example, in the SGR data (Figure 2), there is indeed 1 outlier data in each of the IN and INT groups. This may occur because the estrous cycle that stimulates prolactin can be different in each individual mouse which can affect differences in the growth rate of mammary tumors in rat (Nandi et al., 1995; Schedin et al., 2000). In addition, different levels of stress due to therapy in individual cages could affect differences in the development of mammary tumors in individual rat (Hermes et al., 2009; De la Roca-Chiapas, et al., 2016). We have added this argument in the revision of our article. 2. We have explained the safety of electric field of ECCT to mammry and skin tissues of animal models in the second paragraph of the Introduction as reported in our previous publication. In addition, ECCT has passed the phase I clinical trials for healthy volunteers as stated in the last sentence of the Discussion. The safety of ECCT in normal tissue is determined by the membrane potential of the tissue which is higher than that in cancer cells (Yang and Brackenburry, 2013; Kadir et al., 2018). External electric field exposure cannot penetrate normal cell membranes due to the higher membrane potential (Kirson et al., 2004; Yang and Brackenburry, 2013; Kadir et al., 2018) and only causes rotational motion of the cell organelles (Kirson et al., 2004). On the other hand, external electric field exposure can penetrate cancer cell membranes with lower cell membrane potential, thereby interfering with mitosis (Kirson et al., 2004; Yang and Brackenburry, 2013; Kadir et al., 2018). However, not all normal cells have a higher membrane potential than cancer cells. For example, liver cell has the same membrane potential as MCF-7 cancer cell (Yang and Brackenburry, 2013; Kadir et al., 2018). Even so, we found that liver function was not impaired based on alanine transferase activity and bilirubin levels in blood plasma. The data of the safety of ECCT exposure in rat and healthy volunteers will be published separately this year. 3. We have tried various frequencies (100, 150, and 200 kHz) and intensities (10 Vpp, 18 Vpp and 30 Vpp) of the ECCT electric field in preclinical studies and found that a frequency of 100 kHz and an intensity of 18 Vpp are the optimum electric field exposure for breast cancer therapy. 4. ECCT toxicity data on the liver, kidney and hematological profiles will be published separately this year." } ] } ]	1	https://f1000research.com/articles/10-35
https://f1000research.com/articles/9-1362/v1	23 Nov 20	{ "type": "Brief Report", "title": "Towards establishing extracellular vesicle-associated RNAs as biomarkers for HER2+ breast cancer", "authors": [ "Colin L. Hisey", "Petr Tomek", "Yohanes N.S. Nursalim", "Lawrence W. Chamley", "Euphemia Leung", "Colin L. Hisey", "Petr Tomek", "Yohanes N.S. Nursalim", "Lawrence W. Chamley" ], "abstract": "Extracellular vesicles (EVs) are emerging as key players in breast cancer progression and hold immense promise as cancer biomarkers. However, difficulties in obtaining sufficient quantities of EVs for the identification of potential biomarkers hampers progress in this area. To circumvent this obstacle, we cultured BT-474 breast cancer cells in a two-chambered bioreactor with CDM-HD serum replacement to significantly improve the yield of cancer cell-associated EVs and eliminate bovine EV contamination. Cancer-relevant mRNAs BIRC5 (Survivin) and YBX1, as well as long-noncoding RNAs HOTAIR, ZFAS1, and AGAP2-AS1 were detected in BT-474 EVs by quantitative RT-PCR. Bioinformatics meta-analyses showed that BIRC5 and HOTAIR RNAs were substantially upregulated in breast tumours compared to non-tumour breast tissue, warranting further studies to explore their usefulness as biomarkers in patient EV samples. We envision this effective procedure for obtaining large amounts of cancer-specific EVs will accelerate discovery of EV-associated RNA biomarkers for cancers including HER2+ breast cancer.", "keywords": [ "Extracellular vesicles", "exosomes", "survivin/BIRC5", "long-noncoding RNA", "CELLine bioreactor", "HOTAIR" ], "content": "Introduction\n\nInteractions between tumour and stromal cells sculpt the tumour microenvironment and contribute to cancer malignancy, metastasis and immune evasion. Extracellular vesicles (EVs)1 mediate one of the key intercellular interactions by shuttling biomolecules in micro and nanoscale lipid-enclosed packages. EVs have been associated in many studies with resistance of cancer to chemo or radio therapies2.\n\nEVs contain cargo specific to their parental cell, are very stable, and circulate in blood and other bodily fluids. These properties make EVs prime candidates for cancer detection in liquid biopsies3, either alone or combined with the detection of circulating tumour DNA (ctDNA) or circulating tumour cells (CTCs)4. Upregulation of RNA transcripts including long-noncoding RNA (lncRNA) offers a means for distinguishing EVs originating from tumour and non-tumour cells. LncRNAs are greater than 200 nucleotide-long transcripts constituting two thirds of the transcriptome and they appear to play a critical role in carcinogenesis of many cancers including breast malignancies5. LncRNAs represent promising EV-associated biomarkers but difficulties in producing sufficient amounts of pure cancer associated EVs complicate validation of lncRNA presence in EVs.\n\nHere, we present a simple solution for obtaining high quantities of cancer-associated EVs by culturing the HER2-positive breast cancer cell line BT-474 in a CELLine AD 1000 two-chamber bioreactor flask. The CELLine bioreactor system mimics physiological growth conditions by allowing 3D cell growth on a fibre-mimetic surface, resulting in increases in cell number and EV production6. This strategy allowed us to obtain sufficient EV yields to demonstrate that tumour cells release EVs associated with several potential breast cancer biomarkers.\n\n\nMethods\n\nTo prevent bovine EVs present in foetal calf serum (FCS) from contaminating the cancer-specific EVs, we cultured BT-474 cells from ATCC (ATCC® HTB-20™ ) (seeded at 4.5 × 108 cells/mL) in 15 mL Advanced DMEM/F-12 medium (Gibco, ThermoFisher Scientific, Waltham, USA) supplemented with 2% CDM-HD serum replacement (FiberCell Systems, New Market, USA) in the lower cell chamber of a CELLine AD 1000 bioreactor flask (Argos, Elgin, USA). The same media (150 mL) was used in the upper media chamber but supplemented with 2% FCS (Figure 1A). The dialysis membrane that separates the cell and media compartments allows FCS-specific nutrients <10 kDa but not EVs to pass through and nourish the cells. Every three to four days, the 15 mL of conditioned medium from the cell chamber was harvested for EV isolation, and the media from the upper chamber was replaced.\n\n(A) experimental procedure employed for extracellular vesicle (EV) production, isolation, and purification; (B) transmission electron microscopy image of a small EV; (C,D) hydrodynamic diameter distribution profiles of isolated large and small EVs measured by nanoparticle tracking analysis (NTA) with red vertical lines and blue numbers denote standard deviation and diameters at specific peaks, respectively; (E) EV concentration (empty squares) determined by NTA, and protein levels (filled squares) determined by BCA assay of fractions acquired during separation on a qEV Original size exclusion chromatography (SEC) column; and (F) immunoblot with antibodies specific for HER2, EpCAM and TSG101 proteins. Tetraspanin TSG101 is a loading control. MDA-MB-231 cell lysate serves as the negative control for HER2 and EpCAM proteins. Representative images/data from three independent experiments were shown in B–F.\n\nEVs were isolated using differential centrifugation and size exclusion chromatography (SEC) as outlined in Figure 1. Conditioned medium (15 mL) was first centrifuged at 2,000 x g for 10 min to remove large debris, 10,000 x g for 30 min to isolate large EVs, and 100,000 x g for 70 min to isolate small EVs (Figure 1A). The resulting small EV suspension (in 500 µL PBS) was loaded onto a 35 nm qEV original size exclusion column (Izon, Christchurch, New Zealand), and fractions 7 through 24 were collected using an automated fraction collector (500 µL per fraction). BCA protein quantitation assay (Cat # 23225, Pierce, ThermoFisher Scientific, Waltham, USA) and Nanosight NS300 nanoparticle tracking analysis (NTA; Malvern Panalytical, Malvern, UK) were performed to quantitate protein and particle concentrations in each fraction, respectively. EV concentrations and size distributions were quantified on NTA by recording three 30 seconds videos under low flow conditions. EV-rich fractions (7–11) were pooled, quantified again using NTA and BCA, and concentrated by ultracentrifugation (Avanti, Beckman Coulter, Brea, USA) at 100,000 x g for 70 min.\n\nNegative staining TEM of pooled EV fractions was conducted by adsorption onto Formvar-coated copper grids (Electron Microscopy Sciences, Hatfield, USA) for 2 min, then treated with 2% uranyl acetate for 2 min. Grids were then visualised on a Tecnai G2 Spirit TWIN (FEI, Hillsboro, OR, USA) transmission electron microscope at 120 kV accelerating voltage and images were captured using a Morada digital camera (SIS GmbH, Munster, Germany).\n\nThis procedure was carried out as described previously7. Breast cancer cell lines were grown to log-phase, washed twice with ice-cold PBS, and lysed in an sodium dodecyl sulphate (SDS) lysis buffer [60 mM Tris-HCl (pH 6.8 at 25°C), 2% (w/v) SDS, 10% glycerol]. Proteins (25 μg) were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to PVDF membranes. Membranes were subsequently immunoblotted with antibodies recognising human HER2 (mouse monoclonal, anti-Neu, Santa Cruz, Cat # sc-33684, RRID:AB_627996), human EpCAM (rabbit monoclonal, AbCAM, Cat # ab223582, RRID:AB_2762366), and human TSG101 (rabbit polyclonal, AbCAM, Cat # ab30871, RRID:AB_2208084) and corresponding secondary antibodies. Bound antibodies were visualized using Pierce™ ECL Western Blotting Substrate (ThermoFisher Scientific, Waltham, USA) and the chemiluminescence was measured using a BioRad ChemiDoc MP imaging system (Bio-Rad Laboratories, Inc., Hercules, USA).\n\nTechnical triplicates of Trizol-purified RNA from each experimental condition were reverse transcribed into cDNA using qScript Flex cDNA kit (Cat # 95049, Quantabio, Beverly, USA) primed with equal molar ratio of oligo-dT and random primers according to the manufacturer’s instructions. Quantitative RT-PCR was carried out using SYBR Green MasterMix (Life Technologies, Carlsbad, USA) and gene-specific primers previously validated in the literature (Table 1). These included protein-coding mRNAs EpCAM8, BIRC59, YBX110, GAPDH, and HPRT1, and lncRNAs ZFAS111, HOTAIR12, and AGAP2-AS113.\n\nFor this meta-analysis, the “RSEM expected count (DESeq2 standardized)” dataset was downloaded on 31st March 2020 from the TCGA_GTEx_TARGET cohort included in the UCSC Xena portal and was manually annotated. All data manipulations, plotting and statistical analyses were carried out in R computing environment (v 3.5.3) running in R Studio (v 1.1.414) on a Windows 10 x64 machine. The ggplot2 package (v 3.3.0) was used to render Figure 2B and 2C. Hedges g effect size was calculated using the function cohen.d in the effsize R package (v 0.8.0).\n\n(A) Relative mean mRNA abundance of five protein-coding genes (EpCAM, BIRC5, YBX1, GAPDH, HPRT1) and three long non-coding RNAs (ZFAS1, HOTAIR, AGAP2-AS1) in BT-474 cells and their EVs. Each data point represents the average of three independent experiments (error bars are SEM). (B) Comparison of RNA expression of the gene panel studied in (A) between human tumours and their respective non-tumour tissues deposited in TCGA and GTEx portals. Data were manually classified into 20 different organ categories (y-axis) including 8,867 samples across 28 different cancer types and 6,874 samples across 24 non-tumour tissue types. Colour and area of the circles represent median RNA abundance; darker and larger circles indicate higher RNA expression. (C) Distribution of RNA expression of studied genes in breast tumours and breast non-cancer tissues. Open diamonds denote means of each population. Hedges g effect sizes indicate a number of standard deviations that separates the tumour and non-tumour groups. Hedges g > 0.8 demonstrates large effect size, i.e., difference between the means clearly stands out from the “noise” within the groups.\n\nAn earlier version of this article can be found on bioRxiv (doi: https://doi.org/10.1101/2020.09.27.309252).\n\n\nResults\n\nThe CELLine AD 1000 bioreactor increased the cell density and EV production due to the unique growth surfaces and fluid interactions6,14. In addition, the common issue of contaminating bovine EVs15,16 was avoided by using the serum replacement CDM-HD, which is chemically defined, protein free, and animal component free. From three independent experiments, we obtained an average of 1.9 ± 0.3 × 1011 large EVs of a mean diameter 150 ± 3 nm and 8.5 ± 0.7 × 1011 small EVs of a mean diameter 127 ± 5 nm. Negative-stained transmission electron microscope imaging showed the expected round EV morphology, and NTA size distributions resemble those seen from EVs produced in conventional culture flasks (Figure 1B–D). Low levels of contaminating proteins were observed in fractions 11–24 due to 2% CDM-HD serum replacement instead of the standard 5–10% FCS (Figure 1E). This allowed the accurate quantification of EV-associated protein markers without the concern of contaminating cellular proteins and demonstrated that the small EVs obtained using ultracentrifugation are suitable for RNA analysis.\n\nBoth the BT-474 cell lysates and BT-474 EVs of all sizes and purities isolated contained TSG101, EpCAM, and HER2 proteins (Figure 1F). Consistent with the literature, the triple-negative MDA-MB-231 breast cancer cell line did not express detectable levels of HER2 and EpCAM17. TSG101 is a regulator of the endosomal sorting and trafficking process and is expected to be present in both cells and EVs18. EpCAM is a cell adhesion glycoprotein that has been used extensively as a liquid biopsy marker for several epithelial cancers19, whilst HER2 plays an important role in breast cancer subtyping. Interestingly, HER2-positive EVs appear to increase tumour proliferation and resistance to trastuzumab therapy20.\n\nQuantification of the abundance of several EV-associated RNAs, including protein-coding mRNAs EpCAM, BIRC5, YBX1, GAPDH, and HPRT, as well as lncRNAs ZFAS1, HOTAIR, and AGAP2-AS1, was then performed using qRT-PCR. Despite well-documented differential expression in breast cancer, EpCAM mRNA was not found to be associated with the BT-474 EVs, while BT-474 small EVs were clearly associated with established breast cancer-specific RNAs, including mRNA BIRC5 and lncRNA HOTAIR (Figure 2A).\n\nWe then explored the expression of the identical set of RNAs in 15,741 tumour and non-tumour tissue samples included in The Cancer Genome Atlas (TCGA) and Genotype Tissue Expression (GTEx) databases, respectively. Tumour and non-tumour tissues in all 20 tissues analysed expressed similar levels of YBX1, GAPDH, HPRT1, ZFAS1, and AGAP2-AS1 RNAs. The result indicates a limited use of these RNAs for differentiating tumour and non-tumour EVs. This result is consistent with the canonical “housekeeping” role of HPRT1 and GAPDH and suggests potential use of ZFAS1 and AGAP2-AS1 as housekeeping genes for analyses of lncRNAs in samples including tumour and non-tumour tissues, as well as cultured cells. Of the six candidate biomarkers investigated in this study, only BIRC59, EpCAM8 and lncRNA HOTAIR12 were found to be differentially expressed in a wide range of cancer types including breast cancer (Figure 2B and 2C).\n\n\nDiscussion\n\nWhile EVs hold promise as liquid biopsy targets for breast cancer, efficient production of EVs for molecular characterisation of EV-associated RNA can be challenging using conventional culture systems. In this technical feasibility study, we circumvented this obstacle by culturing BT-474 cells, a commonly used HER2-positive cell line, in a CELLine AD 1000 two-chambered bioreactor, which increased the cell density and EV production due to the unique growth surface and fluid interactions14. In addition, the common issue of contaminating bovine EVs16 was avoided by using the serum replacement CDM-HD, which is chemically defined, protein free, and animal component free. This bioreactor system provided highly enriched EVs in 15 mL of conditioned media, avoiding the sample loss and extra time associated with pre-centrifugation concentrators.We verified that the EVs contained HER2, EpCAM, and TSG101 proteins. Transmission electron microscope imaging also allowed us to be confident that we had truly isolated small and large EVs in accordance with the MISEV guidelines21. We then demonstrated that the BT-474 small EVs were associated with lncRNAs ZFAS1, HOTAIR, and AGAP2-AS, as well as mRNAs BIRC5, YBX1, HPRT, and GAPDH using qRT-PCR.\n\nInterestingly, the cancer-specific EpCAM mRNA was not detected in the small EVs although the EpCAM protein was detectable in the corresponding cell lysates, large EVs, and small EVs. Differential RNA expression in cancer, especially upregulation, has potential to infer a gene’s utility as a biomarker. Our finding indicates that RNAs BIRC5 and HOTAIR are promising EV-biomarkers, particularly in breast cancer, where they are substantially upregulated compared to non-tumour breast tissue. Of interest, EV associated lncRNA HOTAIR was reported to correlate with HER2-positive breast cancer22.\n\nCurrently, proteins dominate the EV biomarker field. However, novel EV-associated breast cancer biomarkers like lncRNAs and other RNAs are being explored more thoroughly to aid in detection and management. RNA biomarkers have higher sensitivity and specificity than proteins because PCR can amplify traces of RNA sequences with high specificity and sensitivity23. Further, it is more economical to detect RNA than protein biomarkers because each protein biomarker requires a specific antibody. These findings demonstrate the efficient production of enriched BT-474 EVs and highlight their unique cargo, especially BIRC5 mRNA and HOTAIR lncRNA. Further studies to determine their clinical significance are warranted.\n\n\nData availability\n\nDRYAD: Towards establishing extracellular vesicle-associated RNAs as biomarkers for HER2+ breast cancer. https://doi.org/10.5061/dryad.jdfn2z39324.\n\nThis project contains the following underlying data:\n\n- Figure_1B_image_hi_mag_25.tif (Raw data for TEM image, high magnification)\n\n- Figure_1B_image_low_mag_24.tif (Raw data for TEM image, low magnification)\n\n- Figure_1C_NTA_Capture_MEV_ExperimentReport.pdf (Raw data from hydrodynamic diameter distribution profiles of isolated large and small EVs measured by nanoparticle tracking analysis (NTA) with red vertical lines and blue numbers denote standard deviation and diameters at specific peaks, respectively)\n\n- Figure_1D_NTA_Capture_SEV_ExperimentReport.pdf (Raw data from hydrodynamic diameter distribution profiles of isolated large and small EVs measured by nanoparticle tracking analysis (NTA) with red vertical lines and blue numbers denote standard deviation and diameters at specific peaks, respectively)\n\n- Figure_1E_qEV_BCA_and_particle_data.xlsx (EV concentration determined by NTA, and protein levels determined by BCA assay of fractions acquired during separation on a qEV Original size exclusion chromatography (SEC) column)\n\n- Figure_1F_raw_not_cropped.pptx (Raw western blot images)\n\n- Figure 2A_RT_qPCR raw data.xlsx (Raw data for RT-qPCR to examine the mRNA expression level of five protein-coding genes (EpCAM, BIRC5, YBX1, GAPDH, HPRT1) and three long non-coding RNAs (ZFAS1, HOTAIR, AGAP2-AS1) in BT-474 cells and their EVs.)\n\n- Figure 2B and C_meta_analysis_rawdata.xlsx (DeSeq2 normalised log2 (x+1) expression values of 10 genes in 8,867 tumours and 6,874 normal tissues downloaded on 31st March 2020 from the UCSC Xena portal)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nThe authors thank Dist. Prof. Bruce Baguley, Drs. Graeme Finlay, Marjan Askarian-Amiri, Herah Hansji and Annette Lasham for helpful discussions.\n\n\nReferences\n\nXu R, Rai A, Chen M, et al.: Extracellular vesicles in cancer - implications for future improvements in cancer care. Nat Rev Clin Oncol. 2018; 15(10): 617–638. PubMed Abstract \| Publisher Full Text\n\nKeklikoglou I, Cianciaruso C, Güç E, et al.: Chemotherapy elicits pro-metastatic extracellular vesicles in breast cancer models. Nat Cell Biol. 2019; 21(2): 190–202. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang W, Xia W, Lv Z, et al.: Liquid Biopsy for Cancer: Circulating Tumor Cells, Circulating Free DNA or Exosomes? Cell Physiol Biochem. 2017; 41(2): 755–768. PubMed Abstract \| Publisher Full Text\n\nChang L, Ni J, Zhu Y, et al.: Liquid biopsy in ovarian cancer: recent advances in circulating extracellular vesicle detection for early diagnosis and monitoring progression. Theranostics. 2019; 9(14): 4130–4140. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZheng Z, Chen M, Xing P, et al.: Increased Expression of Exosomal AGAP2-AS1 (AGAP2 Antisense RNA 1) In Breast Cancer Cells Inhibits Trastuzumab-Induced Cell Cytotoxicity. Med Sci Monit. 2019; 25: 2211–2220. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMitchell JP, Court J, Mason MD, et al.: Increased exosome production from tumour cell cultures using the Integra CELLine Culture System. J Immunol Methods. 2008; 335(1–2): 98–105. PubMed Abstract \| Publisher Full Text\n\nLehrich BM, Liang Y, Khosravi P, et al.: Fetal Bovine Serum-Derived Extracellular Vesicles Persist within Vesicle-Depleted Culture Media. Int J Mol Sci. 2018; 19(11): 3538. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAuber M, Frohlich D, Drechsel O, et al.: Serum-free media supplements carry miRNAs that co-purify with extracellular vesicles. J Extracell Vesicles. 2019; 8(1): 1656042. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchneck H, Gierke B, Uppenkamp F, et al.: EpCAM-Independent Enrichment of Circulating Tumor Cells in Metastatic Breast Cancer. PloS one. 2015; 10(12): e0144535. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWillms E, Johansson HJ, Mäger I, et al.: Cells release subpopulations of exosomes with distinct molecular and biological properties. Sci Rep. 2016; 6: 22519. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHisey CL, Dorayappan KDP, Cohn DE, et al.: Microfluidic affinity separation chip for selective capture and release of label-free ovarian cancer exosomes. Lab Chip. 2018; 18(20): 3144–3153. PubMed Abstract \| Publisher Full Text\n\nJia Y, Chen Y, Wang Q, et al.: Exosome: emerging biomarker in breast cancer. Oncotarget. 2017; 8(25): 41717–41733. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThery C, Witwer KW, Aikawa E, et al.: Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles. 2018; 7(1): 1535750. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang YL, Liu LC, Hung Y, et al.: Long non-coding RNA HOTAIR in circulatory exosomes is correlated with ErbB2/HER2 positivity in breast cancer. Breast. 2019; 46: 64–69. PubMed Abstract \| Publisher Full Text\n\nXi X, Li T, Huang Y, et al.: RNA Biomarkers: Frontier of Precision Medicine for Cancer. Noncoding RNA. 2017; 3(1): 9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHisey CL, Tomek P, Nursalim YNS, et al.: Towards establishing extracellular vesicle-associated RNAs as biomarkers for HER2+ breast cancer. Dryad. Dataset, 2020. http://www.doi.org/10.5061/dryad.jdfn2z393" }	[ { "id": "75230", "date": "07 Dec 2020", "name": "Nagarajan Kannan", "expertise": [ "Reviewer Expertise Breast biobanking", "Mammary gland development", "Breast cancer prevention" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nExtracellular vesicles (EV) are fast emerging as both therapeutic agents and biomarkers. Low yields of EVs in commonly used experimental models have somewhat diminished interest and their further scrutiny. Methodologies to improve EV yield in short term cultures are desirable and much needed for this field.\nIn this brief communication, the authors report a culture method to enrich EV from low EV yielding breast cancer cell line, and using public datasets of tissues to bioinformatically demonstrate that these EVs are associated with RNA species enriched in tumors compared to their respective normal tissues.\nThe EV enrichment method appears to be simple and straightforward, and therefore the applications in cancer field is apparent. I have the following comments for the authors.\nPlease provide the rate of EV production using standard 2D culture method vs your novel bioreactor-based method. It may perhaps appeal to labs working in this area to know the rate of production of EV per ml media per 1million seeded cells per hour in culture (both standard vs bioreactor).\n\nFigures: It is not clear how many times these experiments were repeated or number of samples used. Please state them in the Figure Legends. Figure 1C-D showing hydrodynamic diameter distribution profiles should have intervals in Y-axis.\n\nThe authors should discuss possible drawbacks to this method or choice of cell lines used in this study.\n\nCould you further elaborate on how the TCGA data was analyzed in your methods in order to help reproduce your findings by others.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "75229", "date": "07 Dec 2020", "name": "Tracy K. Hale", "expertise": [ "Reviewer Expertise Cancer and chromatin biology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe identification of extracellular vesicle (EV) biomakers is certainly of interest in breast cancer research. This paper presents methodology to grow and isolate sufficient EVs to enable the investigation of their cargo. This study describes the growth of the breast cancer BT474 cell line in a CELLine AD 1000 bioreactor flask with media containing CDM-HD serum. This allowed the cells to be grown in 3D conditions and large numbers of EVs to be isolated without contamination from bovine EVs.\nFigure 1 could be improved by enlarging region of Fig 1A focusing on the part of the chamber growing the cells and the isolation of EVs.\n\nCould an antibody directed against a histone or tubulin be used in the western blot (Fig 1F) to show that the isolated EVs are not contaminated with any cellular debris?\n\nIn Figure 2A are the differences in HOTAIR (and the others) significant between BT474 cells and EVs? To what p-value?\nThe growth of cells in this system shows that there are differential levels of HOTAIR in the cells vs EVs, and gene expression data is presented to demonstrate the over expression of HOTAIR in tumours.\nIt will be of interest to test the presence of HOTAIR in malignant vs non-malignant EVs, to support the HOTAIR as a breast cancer EV biomarker.\nThe authors discuss the advantages of RNA biomarkers and while a candidate approach is taken here, this ability to isolate sufficient EVs allows them to be used for RNAseq etc.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "75232", "date": "21 Dec 2020", "name": "Bruno M. Simoes", "expertise": [ "Reviewer Expertise Breast cancer biology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors report a novel culture model to isolate and increase the yield of extracellular vesicles (EVs) using a breast cancer cell line. They also claim that some RNA molecules present in EVs are predominantly expressed in tumour tissues compared to their normal tissues’ counterparts.\nMy major concerns are the following:\nIn Figure 1 – Experiments comparing the results with conventional 2D culture system should be shown to support the statements in the paper. And comparison using bovine serum should be shown too.\nUse of specific EV markers is needed to confirm the presence of EVs – see Gonzalez et al, Plos One, 20141.\nUse of at least another cell line is warranted to validate the data and use of primary cells would be ideal to assess potential clinical translation of the data.\nFigure 2A – The rational for selection of this list of EV-associated RNAs is not clear and needs to be defined. Real-time PCR data should be normalized to a reference transcript. Also, it is not clear if these experiments were done with large EVs, small EVs or both? If data corresponds only to small EVs then the experiments should be repeated with large EVs too.\nAuthors state “RNAs BIRC5 and HOTAIR are promising EV-biomarkers” but this is not proven by the data. The housekeeping genes GAPDH and HPRT are also expressed in EVs. Testing the presence of BIRC5 and HOTAIR RNAs in tumour vs non-tumour EVs is warranted.\nNumber of independent experiments and replicates needs to be stated throughout the manuscript.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-1362
https://f1000research.com/articles/10-59/v1	01 Feb 21	{ "type": "Research Article", "title": "Risk factors for major external structural birth defects among children in Kiambu County, Kenya: a case-control study", "authors": [ "George N. Agot", "Marshal M. Mweu", "Joseph K. Wang'ombe", "Marshal M. Mweu", "Joseph K. Wang'ombe" ], "abstract": "Background: Although major external structural birth defects continue to occur globally, the greatest burden is shouldered by resource-constrained countries largely with no surveillance systems. To the best of our knowledge, few studies have been published on the risk factors for these defects in developing countries. The objective of this study was to identify the risk factors for major external structural birth defects among children in Kiambu County, Kenya. Methods: A hospital-based case-control study was used to identify the risk factors for major external structural birth defects in Kiambu County. A structured questionnaire was used to gather information retrospectively on exposure to environmental teratogens, multifactorial inheritance, and sociodemographic-environmental factors during the study participants' last pregnancies. Descriptive analyses (means, standard deviations, medians, and ranges) were used to summarize continuous variables, whereas, categorical variables were summarized as proportions and percentages in frequency tables. Afterward, logistic regression analyses were conducted to estimate the effects of the predictors on major external structural birth defects in the county. Results: From the multivariable analyses, maternal age ≤34 years old, (aOR: 0.41; 95% CI: 0.18-0.91; P=0.03), and preceding siblings with history of birth defects (aOR: 5.21; 95% CI; 1.35-20.12; P =0.02) were identified as the significant predictors of major external structural birth defects. Conclusions: Maternal age ≥35 years old, and siblings with a history of birth defects were identified as the risk factors for major external structural birth defects in Kiambu County, Kenya. This pointed to a need to create awareness among couples against delaying childbearing beyond 35 years of age and the need for clinical genetic services for women of reproductive age with history of births affected by congenital anomalies.", "keywords": [ "Major external structural birth defects", "risk factors", "case-control study", "Kenya" ], "content": "Introduction\n\nWorldwide, an estimated 7.9 million children are born every year with a birth defect, of which approximately 3.3 million die before age five and around 3.2 million could be physically disabled for life1,2. More than 94% of such defects occur in the developing countries where about 95% of these children do not survive beyond childhood1. Birth defects are defined as abnormalities of body structures or functions that develop during the organogenesis period (first-trimester of gestation) and are detectable during pregnancy, at birth, or soon after2,3. These defects may be classified as major when associated with significant adverse health effects requiring medical/surgical care; otherwise, they are described as minor1,2. Alternatively, they can be classified as external when visible at birth or soon after; or internal when advanced medical imaging techniques are required for their detection4–6. Consequently, the phrase ‘major external structural birth defects’ (MESBDs) denotes congenital physical abnormalities that are clinically obvious at birth or soon after which call for medical and/or surgical interventions1,2. The causes of these defects can be classified into three categories: (i) identifiable environmental factors (teratogens/micronutrient deficiencies); (ii) identifiable genetic factors; and (iii) complex genetic and idiopathic environmental factors, described as multifactorial inheritance1,4,7–10. One-third of these causes are attributed to identifiable environmental and genetic factors, whereas the rest are believed to be multifactorial inheritance-related1,4,7–10. Additionally, environmental endowment of women of reproductive age is thought to operate through their socioeconomic and sociodemographic characteristics leading to causes of MESBDs, described as sociodemographic-environmental factors1,4,8–10. Completing more years of education could improve maternal health because educated women are more likely to make informed reproductive health choices than those with low levels of education with a view to improving birth outcomes11–14. Some of the notable maternal decisions include planned pregnancy, preconception folic acid intake in anticipation of conception, and prompt prenatal care11,13,15–20. Maternal occupation could be dependent on educational levels nonetheless occupations such as farming could expose women of reproductive age to teratogenic pesticides21. Organogenesis occurs in the first eight weeks of gestation; however, approximately half of pregnancies are usually unplanned/unintended, thus not recognized until the end of the first trimester1,4,22–24.\n\nTo our knowledge, many studies on the risk factors have been published in developed countries, however, such publications are scanty in developing countries owing to the rarity of the defects, unplanned/unintended pregnancies, and difficulties in identifying these women until the end of the first trimester when the defects have already formed4. To address this gap, this study investigated maternal periconceptional exposure to environmental, sociodemographic-environmental, and multifactorial inheritance-related risks factors for MESBDs in Kiambu County, Kenya. The study assessed: maternal periconceptional exposure to pesticides and teratogenic therapeutic medicines proxied by maternal chronic illnesses (epilepsy and depression); multifactorial inheritance proxied by the history of siblings with birth defects, sex of the last born child, nature of pregnancy, and parity; and sociodemographic-environmental factors consisting of maternal age, level of education, occupation, and adequate prenatal care proxied by gestational age and preconception folic acid intake. The findings of this study could provide great public health opportunities for the formulation of specific treatment strategies, preventive measures, risk-based surveillance systems, and clinical genetic services for the most prevalent MESBDs, regionally and nationally. Consequently, the objective of this study was to identify the risk factors for MESBDs among children in Kiambu County, Kenya.\n\n\nMethods\n\nA hospital-based case-control study was conducted to identify the risk factors for MESBDs. The study participants were recruited as they presented to the child welfare clinics, neonatal/paediatric units and occupational clinics for care during data collection period from May 31st 2018 to and July 31st 2019. A case-control design was the optimal design for this study considering its suitability for the investigation of rare outcomes, as is the case with MESBDs. Even though a population-based design would have been more preferable, the ease of recruiting case and control subjects within the hospital settings disproportionately favoured the hospital-based design. This was an observational study, therefore was reported as per the STROBE guidelines25.\n\nThe study was conducted in 13 hospitals comprising three county referral hospitals (Kiambu, Gatundu, and Thika), eight sub-county hospitals (Karuri, Kihara, Wangige, Nyathuna, Lari, Tigoni, Lussigetti, and Kigumo), and two faith-based hospitals (Presbyterian Church of East Africa Kikuyu Orthopedic and African Inland Church Cure International) situated within Kiambu County, Kenya. Notably, neither population-based or hospital-based surveillance systems for MESBDs existed in the county nor the study hospitals. Nonetheless, cases detected by primary health providers during childbirth and in neonatal care were recorded for the compilation of monthly hospital reports and subsequent entry into the District Health Information System (DHIS). The cases were drawn from Kiambu, Thika, Gatundu, Tigoni, Kikuyu, and Cure hospitals, which provided occupational and rehabilitative health services to children with MESBDs. The controls, on the other hand, were drawn from Kiambu, Gatundu, Thika, Karuri, Kihara, Wangige, Nyathuna, Lari-Rukuma, Tigoni, Lussigetti, and Kigumo hospitals, which provided child welfare services to the under-fives. Kiambu is the second-most densely inhabited county with an estimated population of 2.4 million people out of an estimated national population of 47.5 million26. Its economic mainstay is largely agriculture, comprising tea, coffee, and dairy farming26. Of the county’s total estimated population, approximately 2.2% aged ≥5 years are living with lifelong disabilities26. A study carried out in the county between 2014 and 2018 observed defects of the musculoskeletal system as the most prevalent single system defects followed by central nervous, orofacial clefts genital, ocular, and anal organ defects27.\n\nThe study population consisted of children aged ≤5 years old seeking health services at the study hospitals during the study period spanning from May to July 2019. All children whose mothers consented to participate in the study were recruited.\n\nCases were defined as children aged ≤5 years born with at least one MESBD to resident women of Kiambu County and seeking health care services at the neonatal units, paediatric wards, child welfare clinics and/or occupational therapist clinics of the study hospitals during the three-month study period. The Research Assistants (RAs) liaised with team leads of the departments listed above to identify cases of MESBDs. The team leads had been working in these departments, thus were conversant with the cases seeking services. The team lead invited the mothers of the children who met the case definition to comfortable private rooms within the departments where informed consent was sought and interviews conducted by the RAs. All cases that met this definition and whose carers consented to participate were prospectively recruited into the study until the required sample was attained (see Sample size determination).\n\nControls were children aged ≤5 years born without any forms of birth defects to resident women of Kiambu County and attending routine child-welfare clinics at the study hospitals during the same three-month study period. The Research Assistants liaised with team leads of the child welfare clinics to identify the children without any form of birth defects and were seeking routine immunization, and growth monitoring services. The team leads had been working in these clinics, hence were familiar with most of the under-fives seeking the services. These services are provided between 8.00 am and 5.00 pm from Monday to Friday; the team leads introduced the RAs who then briefed the potential participants on the study objectives. Because of the relatively large number of controls available, they were selected by simple randomization using sealed envelopes upon definition of the sample population and frequency-matched to the cases by the day of presentation.\n\nThe sample size was estimated as per the Kelsey JL et al.28 formula specified for case-control studies as follows: -\n\n\n\n\n\n\n\n\n\n\n\nWhere: n1 is the number of cases and n2 is the number of controls; p1 is the proportion of cases whose caregivers did not begin prenatal care in the first trimester (primary exposure), p2 is the proportion of controls whose care-givers did not begin prenatal care in the first-trimester set at 57%22,23. Remarkably, Zα/2 (1.96) and Zβ (-0.84) are the values specifying the desired two-tailed confidence level (95%) and statistical power (80%), respectively. The odds ratio (OR) for the effect of the primary exposure (cases whose caregivers did not begin prenatal care in the first trimester) was hypothesized to be 3.022,23. The ratio (r) of unexposed to exposed individuals was set at 3, and given the estimates, a total sample size of 408 participants was derived (102 cases, and 306 controls).\n\nBefore data collection, four nursing graduate interns were recruited and trained as RAs on sound interviewing techniques, and information derivation/validation from antenatal care (ANC) booklets. This was to ensure the data collection process spanning three months (May 31st to July 31st, 2019) was conducted in a standardized manner. The ANC booklet contains maternal profile, medical/surgical history, previous pregnancy history, clinical notes, and physical examination findings on ANC visits among others. Maternal profile includes name, age, parity gravidity, height, weight, last menstrual period (LMP), expected date of delivery (EDD) and date of first ANC. Face-to-face structured questionnaires (see Extended data) were administered to the mothers of the study participants by RAs in comfortable secluded rooms within neonatal units and occupational therapy clinics for cases and child welfare clinics for the controls. Data were gathered retrospectively on exposures to environment-teratogens (pesticides and teratogenic medicines proxied by chronic illnesses), multifactorial inheritance (parity, nature of pregnancy, history of siblings with MESBDs and sex of the lastborn child) and sociodemographic-environmental factors (maternal age, education level, occupation, and adequate prenatal care proxied by gestational age and preconception folic acid intake). The predictors were assessed as shown in Table 1.\n\nANC, antenatal care; MESBDs major external structural birth defects.\n\nA conceptual framework depicting the predictor-outcome relationship is displayed in Figure 1. The flow chart of the simple-random systematic sampling strategy is shown in Figure 2.\n\nEthical approval for this study was obtained from the Kenyatta National Hospital [KNH]-University of Nairobi [UoN] Ethics Review Committee [Ref. No: KNH-ERC/A/44]. The purpose of the study was explained to participants and written informed consent was obtained from the mothers of the study subjects before engaging in the study.\n\nConsidering potential biases inherent in case-control studies that were likely to invalidate the study results, deliberate attempts were made to minimize their occurrence. First and foremost, the research assistants were trained on sound interviewing techniques and information derivation/validation from ANC booklets to minimize interviewer and minimize information biases, respectively. In a bid to minimize recall bias, gestational age (weeks) at the first ANC were estimated from the dates of the last menstrual period and dates of the first ANC obtained from the ANC booklets.\n\nFollowing data collection, filled questionnaires were manually checked daily for accuracy and completeness and subsequently entered into a Microsoft Excel spreadsheet (Microsoft Office Professional Plus 2019) by two independent data managers to reduce potential errors. The excel dataset was validated and exported to Stata software version 14.0 (Stata Corporation, Texas, USA) for further cleaning, coding, and analyses. Descriptive analyses (means, medians, standard deviations, and ranges) were used to summarize continuous variables, whereas proportions and percentages for categorical variables were generated and presented in frequency tables. Afterward, the effect of each predictor on the odds of MESBDs was assessed using univariable logistic regression models at a liberal P-value (P≤0.20)29. Gestational age (weeks) at first ANC as a continuous variable was categorized into groups (≤8 weeks and ≥9 weeks) for evaluation in the univariable analyses1,4,22–24. Additionally, parity as a continuous variable was grouped into two groups; primiparous or multiparous categories for assessment in the univariable analyses30,31. However, maternal age as a continuous variable was insignificant in the univariable analyses, thus, recategorized into two groups; ≤34 years, and ≥35 and reassessed for statistical significance; women aged at least 35 years have previously been reported to have an increased likelihood of giving birth to children with MESBDs32. Variables found statistically significant in the univariable analyses were fitted to a multivariable model where a backward stepwise approach was used to eliminate variables from the model at P-value >0.05. To minimize the confounding effects, elimination of non-significant predictors was only considered when their exclusion from the model did not yield more than a 30% change in the effects of the remaining variable29. Two-way interactions were fitted between the remaining variables of the final model and assessed for significance. A Hosmer-Lemeshow test was used to assess the goodness of fit of the logistic model, with a P-value of >0.05 being suggestive of a good fit.\n\n\nResults\n\nA total of 408 study respondents (102 cases and 306 controls) were enrolled in this study. The cases consisted of cleft lip with palate 1 (0.98%), cleft palate 3 (9.94%), clubbed hand 1 (0.98%), club foot 91 (89.22%), hydrocephalus 1 (0.98%), limb defects 4 (3.92%), and persistent cloacal 1 (0.98%)33.\n\nSociodemographic-environment: The median age of the study respondents was 26 years with a mean of 27.31 years (SD=5.73, R; 17-47) (Table 2). The median age of mothers in the case group was 28 years with a mean of 28.73 (SD=5.95, R; 19-47), whereas the median age of mothers in the control group was 26 years with a mean of 26.84 (SD=5.58, R; 17-42) (Table 2). Of the 408 study participants, 184 (45.10%) had attained a secondary level of education; 38 (37.25%) and 146 (47.71%) in the case and control groups, respectively (Table 2). Environmental-teratogens: Of the 408 study respondents, 15 (3.68%) were exposed to farm-sprayed pesticides, of which four (3.92%) were in the case group and 11 (3.59%) were in the control group (Table 2).\n\nSD, standard deviation; R, range.\n\nMultifactorial inheritance: Of the 408 study respondents, 404 (98.77%) had single gestations for the current child, of which 99 (97.06%) and 304 (99.35%) were in the case and control groups, respectively (Table 2). Of the study participants, 15 (3.68%) had given birth to children with birth defect in previous gestations, with 9 (8.82%) in the case group and 6 (1.96%) in the control group (Table 2).\n\nNotably, all the factors assessed for statistical significance in the univariable analyses were associated with MESBDs at P≤0.20; age, education, occupation, sex of the lastborn child, history of siblings with birth defects, preconception folic acid intake, nature of pregnancy, pesticide exposure, chronic illnesses, parity, gestational (age) weeks at first ANC, and ANC beginning eight weeks post-conception (Table 3). Subsequently, these variables were fitted to the multivariable model for the final analysis, except gestational age at first ANC, education, occupation, and prenatal care beginning eight weeks post-conception being distal relative to pesticide exposure and preconception folic acid intake (Figure 1).\n\n*Variables eligible for inclusion in the multivariable model (P≤0.20). CI, confidence interval; MESBD, major external structural birth defect.\n\nIn the multivariable analysis, only maternal age, and history of siblings with MESBDs were shown to be significant predictors at a 5% significance level (Table 4). Compared to women aged ≥35 years old, holding all factors constant, women aged ≤34 years old were 59% less likely to give birth to children with MESBDs (adjusted odds ratio (aOR): 0.41; 95% CI: 0.18-0.91; P=0.03) (Table 4). Additionally, compared to the history of siblings without MESBDs holding all factors constant, siblings with history of MESBDs were 5.21 times likely to have MESBDs (aOR: 5.21; 95%CI; 1.35-20.12; P =0.02) (Table 4).\n\naOR, adjusted odds ratio; CI, confidence interval; MESBD, major external structural birth defect.\n\n\nDiscussion\n\nTo our knowledge, this was the first case-control study conducted to identify the risk factors for MESBDs in the entire country. The study findings corroborated other studies that maternal age greater than 34 years had a strong association with the occurrence of MESBDs34. The study observed that women aged ≤34 years old were 59% less likely to give birth to children with MESBDs compared to those aged over 35-years-old. Older maternal age has been strongly associated with MESBDs such as neural tube defects and orofacial clefs34. Maternal age is a multifaceted risk factor whose mechanisms of action in the occurrence of MESBDs are underpinned by human biology and socio-economic endowment among women of reproductive age. From the biologic standpoint, genetic mutations and accumulation of chromosomal aberrations during the maturation of male germ cells have been attributed to the occurrence of MESBDs1,35,36. The amount of deoxyribonucleic acid damage in sperm of men aged 36–57 is three times that of men <35 years, increasing the likelihood of MESBDs in aging couples35,37. This observation could have been due to environment-physiological interactions as a result of the couples’ socioeconomic endowments1. Similarly, the study results mimiced other research findings across the world and revealed that women whose preceding offspring had MESBDs were at most risk of giving birth to children with MESBDs in the succeeding births. This phenomenon points to the genetic epidemiology implicating multifactorial inheritance in the occurrence of these defects, thus contributing to the global debate on the burden of MESBDs in developing countries8,38–40. Of the 102 cases observed; 1 had cleft lip with palate (0.98%), 3 had cleft palate (9.94%), 1 had clubbed hand (0.98%), 91 had club foot (89.22%), 1 had hydrocephalus (0.98%), 4 had limb defects (3.92%), and 1 had persistent cloacal (0.98%). Defects of a single organ system such as orofacial clefts, talipes, neural tube defects and limb defects have been associated with multifactorial inheritance1,7. Nevertheless, some limitations were inherent in this study; there was a likelihood of differential recall bias among the study respondents; cases were more likely to remember their preconception period owing to the experience of MESBDs in the last birth than the controls, thus could affect their estimates.\n\n\nConclusions\n\nThis study showed that maternal age and history of siblings with MESBDs were the predictors of MESBDs in Kiambu County in Kenya. This pointed to a need to create awareness among couples not to delay childbearing beyond 34 years of age and provision of clinical genetic services such as genetic counseling and screening for families with a history of birth defects. To address this burden, the county should begin by designing and formulating a hospital-based surveillance framework for the most MESBDs to inform specific public health interventions aimed at controlling and preventing specific MESBDs. Additionally, creating public awareness of the risk factors and prevention strategies for these defects through short message during media broadcasts, mobile phone digital platforms, community dialogues, and roadshows. Further, we recommend that similar studies be conducted nationally to inform surveillance, prevention, and control strategies for the most common MESBDs.\n\n\nData availability\n\nFigshare: Risk factors for major external structural birth defects among children in Kiambu County, Kenya: a case-control study. https://doi.org/10.6084/m9.figshare.13614548.v133.\n\nHarvard Dataverse: Risk factors for major external structural birth defects among children in Kiambu County, Kenya: a case-control study. https://doi.org/10.7910/DVN/ZDEOZ5\n\nThis project contains the following extended data:\n\n- 2_mesbds_kmbu_ke_questionnaire.pdf (copy of questionnaire)\n\n- 2_mesbds_kmbu_ke.do (syntax used for analysis)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgments\n\nWe would like to acknowledge the National Commission for Science, Technology and Innovation (Ref. No: NACOSTI/P/19/75586/28325), Kiambu County Commissioner (Ref. No: ED.12 (A)/1/VOL.11/107), and Kiambu County Director of Health, (Ref. No: KIAMBU/HRDU/AUTHO/2019/03/06/AgotGN), for permitting us to carry out this study in the county. We would also like to acknowledge the Medical Superintendents and Directors of the thirteen hospitals for granting us permission and support during data collection. Lastly, we would like to acknowledge the efforts of all data collectors for making this exercise a reality.\n\n\nReferences\n\nChristianson A, Howson CP, Modell B: March of dimes global report on birth defects: The hidden toll of dying and disabled children. White Plains, New York. 2006. Reference Source\n\nWHO: Birth defects surveillance: A manual for programme managers. Geneva, Switzerland. 2014. Reference Source\n\nSever L: Guidelines for Conducting Birth Defects Surveillance. Atlanta: National Birth Defects Prevention Network. Annals of Internal Medicine. 2004. Reference Source\n\nTinker SC, Gilboa S, Reefhuis J, et al.: Challenges in studying modifiable risk factors for birth defects. Curr Epidemiol Rep. 2015; 2(1): 23–30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoorthie S, Blencowe H, Darlison MW, et al.: Estimating the birth prevalence and pregnancy outcomes of congenital malformations worldwide. J Community Genet. 2018; 9(4): 387–96. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nParker SE, Mai CT, Canfield MA, et al.: Updated national birth prevalence estimates for selected birth defects in the United States, 2004– 2006. Birth Defects Res A Clin Mol Teratol. 2010; 88(12): 1008–16. PubMed Abstract \| Publisher Full Text\n\nLucas AO, Stoll BJ, Bale JR: Improving birth outcomes: meeting the challenge in the developing world. National Academies Press; 2003. PubMed Abstract \| Publisher Full Text\n\nKhurmi MS, Gupta M, Chaudhari G: Addressing the public health challenge of birth defects in India. Indian J Child Health. 2014: 95–8. Reference Source\n\nFeldkamp ML, Carey JC, Byrne JLB, et al.: Etiology and clinical presentation of birth defects: population-based study. BMJ. 2017; 357: j2249. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPenchaszadeh VB: Preventing congenital anomalies in developing countries. Community Genet. 2002; 5(1): 61–9. PubMed Abstract \| Publisher Full Text\n\nGrossman M: On the concept of health capital and the demand for health. J Polit Econ. 1972; 80(2): 223–55. Publisher Full Text\n\nOchako R, Fotso JC, Ikamari L, et al.: Utilization of maternal health services among young women in Kenya: insights from the Kenya Demographic and Health Survey, 2003. BMC Pregnancy Childbirth. 2011; 11: 1. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWagstaff A: The demand for health: theory and applications. J Epidemiol Community Health. 1986; 40(1): 1–11. 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PubMed Abstract \| Publisher Full Text\n\nKabubo-Mariara J, Karienyeh MM, Kabubo FM: Child survival and policy options in Kenya: Evidence from demographic and health surveys. Journal of Reviews on Global Economics. 2012; 1: 13–26. Reference Source\n\nBello AI, Acquah AA, Quartey JN, et al.: Knowledge of pregnant women about birth defects. BMC Pregnancy Childbirth. 2013; 13: 45. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTsehay B, Shitie D, Lake A, et al.: Determinants and seasonality of major structural birth defects among newborns delivered at primary and referral hospital of East and West Gojjam zones, Northwest Ethiopia 2017– 2018: case–control study. BMC research notes. 2019; 12(1): 495. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPašková V, Hilscherová K, Bláha L: Teratogenicity and embryotoxicity in aquatic organisms after pesticide exposure and the role of oxidative stress. Rev Environ Contam Toxicol. Springer, 2011; 211: 25–61. PubMed Abstract \| Publisher Full Text\n\nFiner LB, Zolna MR: Shifts in intended and unintended pregnancies in the United States, 2001– 2008. Am J Public Health. 2014; 104 Suppl 1(Suppl 1): S43–S8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFiner LB, Zolna MR: Declines in unintended pregnancy in the United States, 2008– 2011. N Engl J Med. 2016; 374(9): 843–52. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTaye M, Afework M, Fantaye W, et al.: Factors associated with congenital anomalies in Addis Ababa and the Amhara region, Ethiopia: a case-control study. BMC Pediatr. 2018; 18(1): 142. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDa Costa BR, Cevallos M, Altman DG, et al.: Uses and misuses of the STROBE statement: bibliographic study. BMJ open. 2011; 1(1): e000048. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKNBS: 2019 Kenya population and housing census. Nairobi, Kenya: Government Press; 2019. Reference Source\n\nAgot GN, Mweu MM, Wang’ombe JK: Prevalence of major external structural birth defects in Kiambu County, Kenya, 2014-2018. Pan Afr Med J. 2020; 37: 187. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKelsey JL, Whittemore AS, Evans AS, et al.: Methods in observational epidemiology: Monographs in Epidemiology and Biostatistics. 1996. Reference Source\n\nDohoo IR, Martin SW, Stryhn H: Methods in epidemiologic research. 2012. Reference Source\n\nJawad S, Haq IU, Cheema MR: Role of Multiparity in Birth Defects. The Professional Medical Journal. 2017; 24(08): 1241–4. Publisher Full Text\n\nDuong HT, Hoyt AT, Carmichael SL, et al.: Is maternal parity an independent risk factor for birth defects? Birth Defects Res A Clin Mol Teratol. 2012; 94(4): 230–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHollier LM, Leveno KJ, Kelly MA, et al.: Maternal age and malformations in singleton births. Obstet Gynecol. 2000; 96(5 Pt 1): 701–6. PubMed Abstract \| Publisher Full Text\n\nAgot G: Risk factors for major external structural birth defects among children in Kiambu County, Kenya: a case-control study. figshare. Dataset. 2021. http://www.doi.org/10.6084/m9.figshare.13614548.v1\n\nGill SK, Broussard C, Devine O, et al.: Association between maternal age and birth defects of unknown etiology: United States, 1997-2007. Birth Defects Res A Clin Mol Teratol. 2012; 94(12): 1010–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBray I, Gunnell D, Smith GD: Advanced paternal age: How old is too old? J Epidemiol Community Health. 2006; 60(10): 851–3. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBarker S, Chesney D, Miedzybrodzka Z, et al.: Genetics and epidemiology of idiopathic congenital talipes equinovarus. J Pediatr Orthop. 2003; 23(2): 265–72. PubMed Abstract \| Publisher Full Text\n\nYang Q, Wen SW, Leader A, et al.: Paternal age and birth defects: how strong is the association? Hum Reprod. 2007; 22(3): 696–701. PubMed Abstract \| Publisher Full Text\n\nZhou Y, Mao X, Zhou H, et al.: Epidemiology of birth defects based on a birth defect surveillance system in Southern Jiangsu, China, 2014-2018. J Matern Fetal Neonatal Med. 2020; 1–7. PubMed Abstract \| Publisher Full Text\n\nXie D, Yang T, Liu Z, et al.: Epidemiology of birth defects based on a birth defect surveillance system from 2005 to 2014 in Hunan Province, China. PLoS One. 2016; 11(1): e0147280. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWellesley D, Boyd P, Dolk H, et al.: An aetiological classification of birth defects for epidemiological research. J Med Genet. 2005; 42(1): 54–7. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "81051", "date": "16 Mar 2021", "name": "Yoseph Worku", "expertise": [ "Reviewer Expertise Public Health and Epidemiology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIt is interesting, technically sound and intelligibly written manuscript. There are minor points to be improved.\n\nIn the abstract, the results and the conclusions part should show consistent interpretation and conclusion. The conclusion should base on age < 35 years. The reference is Age > 35 years (would have been better to take the <35 years as a reference). While presenting the classes, since it is dichotomized, it is better to show a common number as a margin of the classes . E.g. <35 and > 35 OR < 9 and > 9.\n\nThe Conceptual Framework should reflect the classifications of the risk factors presented in the Introduction. Some of the variables need to be regrouped in themes and the Framework should be redesigned accordingly.\nSome of the variables need to be defined. E.g. Pesticide exposure, chronic illness,... Check the Sample Size Determination part - the Epi Info calculation does not show the same number.\n\nIn the univariable analyses, why p-values for the reference categories are included? The discussion is a bit shallow. Comparison with more literatures, more in-depth look in to the implications and significances of the findings, and addressing also key relevant factors without significant association in the current study can improve the Discussion part. The paternal age was also mentioned as key factor in previous studies but not assessed in the current study. Why? There are also other possible limitations not mentioned. E.g. survivor bias and not controlling for some relevant variables in the multivariable analysis like the paternal age.\n\nThe conclusion is a bit beyond the scope of the study. E.g. awareness level of couples or the community is not assessed. Detailed and in-depth discussion by citing other relevant literatures can help readers to better understand the situation and to deduce more appropriate conclusions.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6561", "date": "30 Apr 2021", "name": "George Agot", "role": "Author Response", "response": "Abstract: Conclusions have been aligned to the study results and interpretations. Maternal age <35 years has been used as the reference category, and presented as <35 years, and >= 35 years, however it was not included in the inclusion because it was no longer associated with dependent variable in the univariable analyses. Gestational age: Gestational age has also been presented as <9 weeks, and >=9 weeks. Pesticides exposure: Exposure redefined as maternal exposure to farm-sprayed pesticides before conception. Chronic illnesses: Chronic illnesses were used as a proxy for measuring maternal use of teratogenic therapeutic agents for chronic conditions such as epilepsy, depression, hypertension and diabetes mellitus. In this respect, it was not used as a measure for particular chronic illnesses. Conceptual framework: Redesigned to reflect the three classes of major external structural birth defects described in the study introduction. Univariable analyses: P-values for binomial variables were deleted, whereas likelihood ratio test (LRT) was performed for nominal variables/variables with more than two categories to estimate the associated P-values for each variable. Discussion: Discussion of the significant variables was improved to include their implications, and significance. Variables that showed no associations were also explained with reference to other studies. Paternal age: Paternal age was introduced in the model, however it showed no association with the defects in the univariable analyses. Nevertheless, it was controlled for in the multivariable, but still showed no association. Further, because of potential collinearity with materteral age, paternal age was controlled for without maternal age in the multivariable analyses, however, still showed no association with birth defects. Limitations of the study: Survivor bias was included as a limitation of this study because some of these defects for example neural tube defects are potentially fatal. Conclusions: Conclusions were aligned to the study findings and interpretations." }, { "c_id": "6562", "date": "30 Apr 2021", "name": "George Agot", "role": "Author Response", "response": "Sample size determination: The sample computation was reconfirmed and found consistent with formula provided, however the hypothesized odds ratio is 2.0 (universally accepted, and not 3.0 as presented. This typing error has since been corrected." } ] }, { "id": "81531", "date": "07 Apr 2021", "name": "Marcia L. Feldkamp", "expertise": [ "Reviewer Expertise Epidemiology", "birth defects" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe investigators present a hospital-based case-control study conducted in Kiambu County, Kenya. The paper is well-written and the methodology easy to follow that was used to investigate risk factors for major external structural malformations. The investigators are to be commended for evaluating risk factors for structural malformations in a developing country. This is an important step toward understanding potential risk factors for the ultimate goal of primary prevention. I have a few suggestions for the investigators to consider that may strengthen the paper.\nMethods:\nThere are inconsistencies with the specified time period of data collection/enrollment of subjects: “May 31, 2018 to and July 31, 2019”; “May to July 2019”; “three-month study period”.\n\nExposure time period should be clarified for pesticides and how the exposure determined. The investigators should also consider investigating maternal tobacco use, pre-gestational diabetes (specifically), and periconception infections.\n\nThe investigators conducted a sample size calculation based on all cases. The challenge with this idea is that birth defects are a very heterogeneous group and lumping them altogether suggests that their risk factors are similar. Unfortunately, this is not the case.\nResults:\nTable 3: no need to have a p value listed for the reference group.\n\nSince the investigators report an increased risk for maternal age >35 years, did any of the cases have a chromosomal etiology?\n\nDid affected siblings have the same type of birth defect?\nDiscussion - Limitations: small sample size, based on combining several different types of birth defects.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "6563", "date": "30 Apr 2021", "name": "George Agot", "role": "Author Response", "response": "Methods 1. Data collection period: This has been corrected to imply a three-month data collection period from May 31, 2019 to July 31, 2019. It was a typing error, but has since been corrected. 2. Exposure time period for pesticides: This variable has been defined as maternal exposure to farm-sprayed pesticides at conception. Increased likelihood of maternal exposure to pesticides was considered because agriculture is the economic mainstay in the county. Maternal tobacco use: -This variable was assessed as a variable of interest, however was dropped because only 2 out 0f 408 respondents reported its use. Pre-gestational diabetes: - This was not assessed as a variable of interest, however it was alluded to and assessed among other chronic illnesses as a proxy for measuring maternal use of teratogenic therapeutic agents for conditions such as epilepsy, depression, hypertension and diabetes mellitus. Infections: - Infection is indeed an important predictor for birth defects, however, it was beyond the scope of this study since data were gathered retrospectively. 3. Sample size calculation: Birth defects are largely heterogeneous in their etiology, however the defects were lumped together for sample size calculation because of the extreme rarity of these defects coupled with unavailability of hospital-based/population-based surveillance programs in the county. Nevertheless, this has been cited as a limitation of the study. Results 1. Table 3: P-values for binomial variables were deleted, whereas likelihood ratio test (LRT) was performed for nominal variables/variables with more than two categories to estimate the associated P-values for each variable. 2. Maternal age: The reference category for maternal age was changed to <35 years, but showed no association in the univariable analysis. Nonetheless, the study results were suggestive of chromosomal etiology because some cases were reported to occur with down syndrome, whereas autism was also reported by control subjects as a defect in the previous births. 3. Siblings with same types of birth defects: Yes this was observed in the study. A recurrence of clubfoot was reported by some case subjects, whereas other types of birth defects were reported by case subjects to have occurred with clubfoot Discussion: Small sample sample based on combined several different types of birth defects has been cited as a limitation of this study." } ] }, { "id": "81820", "date": "09 Apr 2021", "name": "Rogath Kishimba", "expertise": [ "Reviewer Expertise Maternal and Child Health Epidemiologist" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a good and important research area for newborn health particularly now when a lot has been done on infectious unlike non infectious diseases. We have observed a great decrease of infant mortality given the available maternal and newborn interventions on infectious diseases. A higher contribution of non infectious disease particularly birth defects may be observed on neonatal and infant mortality with time. Below are my inputs and comments regarding this study;\nThe title, abstract and introduction are well written.\n\nCurrent citations were used.\n\nThe study design is appropriate however selection of cases was not appropriate given the study title and objectives. It can be admitted as one of study limitation.\n\nCases were sampled from child welfare clinics, neonatal/pediatric units, occupational and rehabilitation clinics. All these data sources represent survivors of MESBDs and most probably non fatal MESBDs. It is difficult to get fatal MESBDs like neural tube defects (NTDs) cases from this subpopulation as majority will not survive to meet them in rehabilitation clinics.\n\nThe ascertainment period from the case definition is too high (5years and below). This may lead to potential recall bias as it will be very difficult for a mother to remember what happened in her pregnancy in the 3-4 years ago. Again may lead to recruitment of survivors and non fatal MESBDs cases. This could be mitigated for at least to consider/restrict enrolment into the study for children below 1 or 2 years only.\n\nI understand well that the data sources were the above mentioned clinics which are complimented by the ANC booklets. However the methodology section again mentioned about DHIS and I was wondering whether it was also another data source which was used. It needs clarity for the reader to well understand sources of data for this study.\n\nThe methodology section need more clarity on maternal age. Is it the age of the mother during conception of the referred case? or the age of the mother during the data collection? It is also very important to define \"residence\" as it has implication on maternal exposures. The residence is important during conception and antenatal period. This is the period when environmental exposures can have impact on the unborn child. There is no any significance of considering residence post delivery.\n\nSample size calculation is Ok. However you can not estimate proportion of controls (p2) using a study with a different objectives from your intended study.\n\nThe hypothesized odds ratio for the effect of the primary exposure is too high. This is the risk which you allow to be detected in your study. At least you can allow a minimal risk of odds ratio between 1.5 and 2.\n\nResults were well written however there is a need to your interpretation and conclusion to reflect your exact results. If the maternal age ≤34 years was found to be protective does not mean the maternal age ≥35 years is a risk. Remember this age category was your reference. If you want to refer the age category ≥35 years then make the other category \" ≤34 years\" a reference in your logistic regression analysis. Otherwise I advise to interpret and make conclusion exactly as what you found in your result section.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "6566", "date": "30 Apr 2021", "name": "George Agot", "role": "Author Response", "response": "Sampling of cases: Survivor bias has since been included as a limitation of this study because some of these defects for example neural tube defects are potentially fatal, yet such data could not be gathered because we adopted a retrospective approach to gather the information. The ascertain period for cases: It is true this period could potentially attract recall bias. This period was preferred considering the defects are extremely rare coupled with unavailability of surveillance systems in the county. Even though we preferred <5 years, we encountered children aged <4 years and approximately 78% were aged <2 years, thus, this limitation was somehow reduced by default. DHIS: This was not used as a source of data in this study, however was intended to inform the readers on how data on birth defects are routinely gathered in the county in the absence of surveillance systems. Maternal age: Maternal age was defined as maternal age at conception. Residence: Residence was defined as maternal place of residence at conception, thus introduced in the model and controlled for in multivariable logistic analysis. Sample size calculation: The choice of literature for p2 was informed by maternal peri-conceptional period, especially 8 weeks of gestation when defects are expected to have formed yet most women do not plan their pregnancies, thus making it difficult for health care workers to identify these women in good time to effectively implement public health preventive measures before the defects form. Majority of such women attend first ANC at the end of second trimester of gestation when the defects have already formed. Hypothesized odds ratio: Hypothesized odds ratio for the effects of the primary exposure is actually 2.0 (universally accepted) and not 3.0 as presented in the manuscript. This was a tying error and has been corrected. Results: The reference category for maternal age was changed to <35 years, but showed no association in the univariable analysis. Conclusions: The conclusions have been aligned to the study findings and interpretations." } ] } ]	1	https://f1000research.com/articles/10-59
https://f1000research.com/articles/10-335/v1	30 Apr 21	{ "type": "Research Article", "title": "The existence and characteristics of rats and shrews in endemic leptospirosis areas and types of ectoparasites: a case study of West Jakarta, Indonesia", "authors": [ "Dewi Susanna", "Rusyda Ihwani Tantia Nova", "Laura Rozek", "Rusyda Ihwani Tantia Nova", "Laura Rozek" ], "abstract": "Background: This study aimed to determine the presence and species of the rats and shrews that can potentially cause leptospirosis in West Jakarta, Indonesia, and the species of ectoparasites found in them. Methods: The research was a descriptive study employing a cross-sectional approach. The study population was all species of rats and shrews in the region and the sample collection technique used was purposive sampling. The traps were installed in the homes of respondents who had suffered from leptospirosis and their closest neighbors, with a total of 521 traps. Leptospirosis data based on secondary data was obtained from West Jakarta Health Office (2016-August 2019). The technique for catching rats involved using humane live traps, while the identification of the rats and ectoparasites was done in the laboratory. Results: It was found that more rats were caught in Cengkareng Timur sub-district, Cengkareng District, with a percentage of 14.8%, while the least in Duri Kepa, Kapuk, Kedaung Kali Angke and Kedoya Utara with a percentage of 3.7%. The rats were mostly found in East Cengkareng Sub-District, with the most common type being Rattus rattus (74.1 %) and the least Suncus murinus (11.1%); more male rats were caught (66.7%) than female (33.3%). The type of ectoparasite found in the rats was fleas. Xenopsylla cheopis was the most common type, at 83.3% and more fleas were male, at 66.7%. The most common rat species was Rattus rattus. The ectoparasite most commonly found in them was the female flea Xenopsylla cheopis. Conclusions: Rattus rattus and Xenopsylla cheopis were found in an East Cengkareng sub-district. Surveys, monitoring, and control of rats and ectoparasites are essential for the preparedness and development of an early warning system of possible diseases that they can cause.", "keywords": [ "species", "rats", "ectoparasite", "shrew" ], "content": "Introduction\n\nRodents such as rats and shrews can carry various bacteria and viruses that can cause infections in humans. They are thought to be a reservoir of 30% of zoonotic pathogens, including several viruses, bacteria, and parasites. Rats are one of the most important components of an ecosystem. Their presence is very widespread, representing 40% of all mammal species (Churakov et al., 2010). They can be useful as food for some mammals and predatory birds (Tobin & Fall, n.d.), but their presence in the ecosystem can cause various losses in sectors such as agriculture and health (Tobin & Fall, n.d.). Rats are a reservoir of various diseases, and as animals that cannot be separated from human life, they can cause various health problems in humans and pets and other wildlife (Tobin & Fall, n.d.). They are also an important host for ectoparasites and have a close relationship, such as lice, fleas, and mites (Kiffner, Vor, Hagedorn, Niedrig, & Rühe, 2011). They can produce a variety of associations influenced by the host and parasite species and the biotic and abiotic environment (Buchholz & Dick, 2017). The rate of rat ectoparasite infestation can reach 66.6%, and ectoparasites are vectors for various diseases which can cause health problems (Zendehfili, Zahirnia, Maghsood, Khanjani, & Fallah, 2015).\n\nLeptospirosis can cause death, with the case fatality rate in humans of 5-30% (CDC, 2018). It is a disease caused by Leptospira bacteria (World Health Organization, 2003). Leptospira interrogans and Leptospira borgpetersenni are present in many populations of rats and have been confirmed to cause leptospirosis in humans (Cosson et al., 2014). The disease can be transmitted through water or soil contaminated by the urine of rats infected with Leptospira bacteria and direct contact with infected animals (Centers for Disease Control and Prevention, 2018; Haddis, 2004; World Health Organization, 2003). Leptospira bacteria can enter the human body through mucous membranes, wounds, or blisters on the skin (Centers for Disease Control and Prevention, 2018; Haddis, 2004; World Health Organization, 2003). In several previous studies, it has been found that Rattus norvegicus is the rat species that is the main reservoir of Leptospira bacteria (Marcos Tucunduva de Faria et al., 2013; Pui, Bilung, Apun, & Su’ut, 2017).\n\nEnvironmental conditions and their habitat greatly influence the presence of rats; each species has a different habitat. Mus musculus is a rat species that like to live in homes, outbuildings, and shops (Global Invasive Species Database, 2015). Rattus rattus is spread in forests and can also live in and around buildings, both underground and above ground (Csurhes, 2012). Rattus norvegicus is a species that is widespread in places such as sewers, agricultural and horticultural land, grasslands, and the interior part of the region (Hausser & de Roguin, 1995). The rat population will continue to increase to the level of capacity accommodated by their habitat (Jackson, 1972). Moreover, the rat population will also greatly depend on the availability of food and predators' presence in their habitat.\n\nWest Jakarta is one of the five administrative cities in the Special Capital Region of Jakarta, Indonesia; its center is in Kembangan. West Jakarta has an area of 129.54 km2 and eight subdistricts, namely Taman Sari, Tambora, Kembangan, Kalideres, Cengkareng, Palmerah, Kebun Jeruk, and Grogol Petamburan Districts (Badan Pusat Statistik Administrasi Jakarta Barat, 2019). West Jakarta is one of the areas with 70 cases from January 2016 to August 2019 (Suku Dinas Kesehatan Jakarta Barat, 2019). The population density in West Jakarta in 2018 was 19,757 inhabitants/km2, with an average population per household of four (Badan Pusat Statistik Administrasi Jakarta Barat, 2019). This means that West Jakarta is a densely populated area, one of the conditions that rats highly favor. This research aims to establish the presence and species of the rats and shrews that can potentially cause leptospirosis in West Jakarta and the species of ectoparasite found in them. The research could be used as the basis for an early alert system for various diseases that rats can carry, and also as a preliminary study to ascertain which rat species are the main reservoirs of leptospirosis in West Jakarta.\n\n\nMethods\n\nThis research describes the presence and species of the rats and shrews in an endemic leptospirosis area in West Jakarta, employing a cross-sectional approach. The study population comprised all species of rats and shrews in the region. The research was conducted in December 2019 and consisted of all people diagnosed with leptospirosis, which referred to the doctor's diagnosis results through clinical reports and laboratory tests that were reported and recorded in the West's work area Jakarta Health Office from January 2016 to August 2019. The sample collection technique used was purposive sampling, involving the installation of rat traps at the homes of participants who had suffered from leptospirosis and their closest neighbors.\n\nThe rodents were caught using a live trap. This live trap has no brand made from wire 34 cm length, 20 width, and 15 heights. Each house had two live traps installed over two consecutive days with a total of 128 houses (16 houses in Kembangan District, 10 houses in Grogol Petamburan District, 24 houses in Cengkareng District, 18 houses in Kebun Jeruk District, 30 houses in Kalideres District, 12 houses in Palmerah District, and 18 houses in Tambora District) and 512 traps (64 live traps in Kembangan District, 40 live traps in Grogol Petamburan District, 96 live traps in Cengkareng District, 72 live traps in Kebun Jeruk District, 120 live traps in Kalideres District, 48 live traps in Palmerah District, and 72 live traps in Tambora District). The bait used was salted fish, which was changed every day during the capture process. In each house, as many as two traps were installed in the place where small mammals are suspected of passing by, evidenced by signs of rodents such as footprints, rat droppings, the smell of rodents, bite marks, digs/earthen holes, and the sound of small mammals. The traps were installed in the afternoon between 15.00 and 17.00 WIB, and the rodents were collected in the morning between 07.00 and 09.00. The traps managed to ensnare rats on the first day were taken and replaced with new ones. Trapped rodents were then labeled by name, serial number, head of families (head of household), district, community neighborhood (RW), neighborhood unit (RT), date, and the day the rat was trapped. The rats caught on the first and second days were collected and stored in the respondent's home to be collected the next day by the researchers. The rodents were put into white sacks and their traps and then taken to the Tanjung Priok Class I Port Health Office (KKP) for identification.\n\nThe rats were put into an airtight plastic bag and anesthetized using chloroform with a dose of 5 – 10 mL. The chloroform was poured into cotton, and the cotton was put into the plastic bag. After the chloroform was added, the rats were left for 10 to 15 minutes until the rats and shrews passed out or died. All rats died from the chloroform dose. Identifying the rats was performed by using external morphological signs such as body length, tail length, back foot length, ear length, head length, mammae, and body weight. Besides, the hair color, type, and size were also considered and then matched with the rat identification key (Badan Penelitian dan Pengembangan Kesehatan & Kementerian Kesehatan Republik Indonesia, 2016). After the identification, the rats and shrews were put in plastic bags and buried with a depth of 2,5 m. Before being buried, the plastic bag was disinfected using alcohol 70%. It will handle by Tanjung Priok Class I Port Health Office or Kantor Kesehatan Pelabuhan (KKP) according to existing procedures (Direktorat Jenderal Pencegahan dan Pengendalian Penyakit Tular Vektor dan Zoonotik, 2019).\n\nThe rats that had fainted or died were placed in a white tray, then combed with a flea comb. Ectoparasites that fell into the tray were taken using tweezers, placed into a bottle of 70% alcohol, then labeled according to the area where they were caught. Ectoparasites with hard skin such as fleas were first soaked in 10% KOH solution for 24 hours. They were then put into six Petri dishes, previously filled with alcohol, aqua dest, and xylol solution (6th Petri dish) alternately with a transfer time span of 2 minutes for each Petri dish. The fleas were then placed inside a glass object and covered with a glass deck. Each part of the edge of the glass deck was glued to close tightly to the glass object. The ectoparasites were then examined under a microscope and matched to the ectoparasite identification key (Mathison & Pritt, 2014) in Tanjung Priok Class I Port Health Office, Jakarta.\n\nAll the procedures complied with the National Research Committee's ethical standards. The study was approved by the Research and Community Engagement Ethical Committee of the Faculty of Public Health, Universitas Indonesia No. 650/UN2.F10/PPM.00.02/2019. All efforts were made to ameliorate harm to the animals by ensuring the animal welfare by following the all procedures in accordance with the technical instructions for rats surveillance laboratory-based and the guidance for rats and mice control (Kementerian Kesehatan Republik Indonesia, 2015).\n\n\nResults\n\nBased on the results of the study, it was found that more rats were caught in Cengkareng Timur sub-district, Cengkareng District, with a percentage of 14.8% of the total (Table 1) (Susanna, Nova & Rozek, 2021). Based on the result, it can be seen that the rat species most commonly found in the West Jakarta area was Rattus rattus (74.1%), Rattus norvegicus (14.8), Suncus murinus (11.1%), and more male rats were caught, equal to 66.7% (female 33.3%) (Table 2). The only type of ectoparasite found in the rats in the West Jakarta area was the flea (Table 3). Xenopshilla cheopis was the most common type (83.3%) and Xenopsylla astia (16.7%). More fleas were male (66.7%) than female (33.3%).\n\n* The region was found more rats and Shrews.\n\n\nDiscussion\n\nCengkareng district is the administrative area of West Jakarta with the highest population, comprising 514,416 people (Badan Pusat Statistik Kota Jakarta Barat, 2019). It has 26.54 km2 and includes six sub-Districts; Cengkareng Timur sub-District is one of the densely populated areas in Cengkareng district (Badan Pusat Statistik Kota Jakarta Barat, 2019). Based on the research results, rats are most commonly found in the East Cengkareng sub-District. This study found different results to previous studies, which found that rats were more commonly found in agricultural areas (58.0%) (Munõz-Zanzi, Mason, Encina, Gonzalez, & Berg, 2014). These different results could be caused by regional differences and the categories of the research areas. Previous studies [25] have researched three locations, namely agricultural areas, rural areas, and slums, while this study was only conducted in West Jakarta, without categorizing the area.\n\nThe presence of rats in an area depends on ecology, vegetation, food availability, and predators' presence. Also, the presence of rats also correlates with the number of tree species; their number will tend to increase in areas with high tree diversity (Madden et al., 2019). However, in this study, the level of tree diversity did not affect rats' presence, as the habitat of those caught was in homes and gutters. Moreover, Cengkareng Timur sub-district is not an area that has a diversity of trees. Seasons also have an important influence on rats' presence; house or commensal rats will be more common in the dry season (Panti-May, Hernández-Betancourt, Ruíz-Piña, & Medina-Peralta, 2012). In this study, the rats were found in densely populated areas, and the process of catching them took place in the dry season so that more would be caught. In the dry season, the availability of rat food is higher, especially leftovers from processed household products, as well as from some home industries in the Eastern Cengkareng sub-district.\n\nIn this study, the rat species most commonly found was Rattus rattus (black rats). This species was commonly found because catching the rats and setting traps was commonly found in participants' homes. Rattus rattus is a species whose habitat is forests and homes (Csurhes, 2012). The species can also be found in natural and semi-natural habitats (The ICUN Red List, 2017). It is an arboreal animal that can climb. Rattus rattus favors lowlands that are less than 250 meters above sea level (Isnaini, 2008). The environmental conditions greatly affect the presence of Rattus rattus in the ecosystem, for example, the availability of food sources. Food, organic waste that is not stored or properly disposed of, gardens that are not well managed; and the presence of pets and livestock can affect the number of rats, including the Rattus rattus species (Feng & Himsworth, 2014).\n\nThe presence of rats in the community environment can cause various health problems, one of which is leptospirosis, a disease caused by Leptospira bacteria (Centers for Disease Control and Prevention, 2018; Haake, David A, & Levett, 2015; World Health Organization, 2003). Rats are the most important reservoir of Leptospira bacteria. Previous studies have found a similarity between the PFGE pattern and gyrB sequence in Leptospira bacteria isolated from humans and rats in Luzon, Philippines (Villanueva et al., 2014). This indicates that rats infected with Leptospira bacteria can cause leptospirosis in humans (Villanueva et al., 2014). The prevalence rate of Leptospira bacteria in each rat species is different. Based on the results of previous studies, it is known that Leptospira spp positively infects up to 17.8% of the Rattus rattus species., 30.3% of the Rattus norvegicus species, 10.9% of the Rattus exulans species, 19.3% of the Rattus argentiventer species, and 3.4% of the Rattus tanezumi species (Id, Shiokawa, & Id, 2019; Koizumi et al., 2009). From these results, it is clear that Rattus norvegicus is the most infected species with the Leptospira bacteria, which causes leptospirosis in humans. However, to establish which species of rats are most responsible for carrying Leptospira bacteria and causing leptospirosis, further research is needed.\n\nIn addition to the presence of rats, ectoparasites can also cause other health problems for humans. One disease that can be caused by rat ectoparasites (fleas) is pes (plague) (Illinois Department of Public Health, n.d.; Nurisa, 2005). Based on the study findings, out of the 27 rats caught, some had ectoparasites such as fleas. The most common type of flea was Xenopsylla cheopis (male). The results of this study are in line with those of previous study by Maulana et al., which also found that several species of rats carried ectoparasites, with the most commonly found type being Xenopsylla cheopis (81.1%) (D, 2012). In another study by Kia et al, it was also found that one of several fleas found in Rattus rattus and Rattus norvegicus was related to the transmission of plague. The most abundant ectoparasite (88.7%) in Bandar Abbas, Southern Iran, was Xenopsylla, found in Rattus norvegicus (Kia et al., 2009).\n\nA previous study by Ristiyanto et al. had also found that female Rattus tanezumi had more ectoparasites than males, while more ectoparasites were in male Rattus exulans rats than in female ones (Ristiyanto, Mulyono, Agustina, Yuliadi, & Muhidin, 2011). Riyanto also found that the most common type of flea found in house rats was Xenopsylla cheopis. This suggests the potential for disease caused by rats and their ectoparasites (Riyanto, 2019). However, not all rats will have ectoparasites, as many aspects can influence their presence. One aspect that can affect the presence of ectoparasites in rats is the season. In the summer, due to dry and hot weather, rats and ectoparasites' presence is low (Alahmed & Al-Dawood, 2001). The presence of rat ectoparasites that can potentially cause health problems leading to high mortality and morbidity must be controlled properly.\n\n\nConclusion\n\nRats were mostly found in Cengkareng district, specifically in the East Cengkareng Sub-District, one of the districts with a high incidence of leptospirosis in West Jakarta. The most common rat species is Rattus rattus. The ectoparasite most commonly found in rats is the male flea Xenopsylla cheopis. Surveys, monitoring, and control of rats and ectoparasites are essential for the preparedness and development of an early warning system of possible diseases that they can cause. Future research should include data about the relative abundance index of trapping, a map of the trapped area, and information about the house/region.\n\n\nData availability\n\nDryad: Ectoparasites in rats and shrews data related to leptospirosis in West Jakarta. https://doi.org/10.5061/dryad.t4b8gtj18 (Susanna, Nova & Rozek, 2021).\n\nThis project contains the following underlying data:\n\n- Data 1. sav (dataset containing the region, name of species Rattus, gender, body length, tail length, rear length, feet length, earlobe length, head length, mamae, and weight.)\n\n- Data 2. sav (dataset containing species of ectoparasite and gender)\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgments\n\nThe authors would like to thank the public health center staff in West Jakarta who assisted the authors during the data collection.\n\n\nReferences\n\nAlahmed AM, Al-Dawood AS: Rodents and their ectoparasites in Wadi Hanifah, Riyadh City, Saudi Arabia. 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Jakarta: Direktorat Pencegahan dan Pengendalian Penyakit Tular Vektor dan Zoonotik; 2019.\n\nFeng AYT, Himsworth CG: The secret life of the city rat: A review of the ecology of urban Norway and black rats (Rattus norvegicus and Rattus rattus). Urban Ecosystems. 2014; 17(1): 149–162. Publisher Full Text\n\nGlobal Invasive Species Database: Species Profile: Mus Musculus. 2015.\n\nHaake DA, Levett PN: Leptospirosis in Human. PubMed Central. 2015; 387. Publisher Full Text\n\nHaddis A: Control of Insects and Rodents For Health Extension Workers, (November), 97 pp. 2004.\n\nHausser J, de Roguin L: Rattus norvegicus. In: Säugetiere der Schweiz/ Mammifères de la Suisse/Mammiferi della Svizzera. 1995; (pp. 283–287. Birkhäuser Basel. Publisher Full Text\n\nId KB, Shiokawa K, Id SR: Leptospira infection in rats: A literature review of global prevalence and distribution. 2019; 1–24.\n\nIllinois Department of Public Health:Reducing the Risk of Human Infection from Pet Rodents How to Reduce the Risk of Infection, (Lcmv).n.d.. Reference Source\n\nIsnaini T: Tikus rumah. Balaba: Jurnal Litbang Pengendalian Penyakit Bersumber Binatang Banjarnegara. 2008; 006(01): 20.\n\nJackson WB: Biological and behavioural studies of rodents as a basis for control.1972; 281–286. PubMed Abstract \| Free Full Text\n\nKia E, Moghddas-Sani H, Hassanpoor H, et al.: Ectoparasites of rodents captured in Bandar Abbas, Southern Iran. Iran J Arthropod Borne Dis. 2009; 3(2): 44–49. Reference SourcePubMed Abstract \| Free Full Text\n\nKiffner C, Vor T, Hagedorn P, et al.: Factors affecting patterns of tick parasitism on forest rodents in tick-borne encephalitis risk areas, Germany. Parasitol Res. 2011; 108(2): 323–335. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKoizumi N, Muto M, Tanikawa T, et al.: Human leptospirosis cases and the prevalence of rats harbouring Leptospira interrogans in urban areas of Tokyo, Japan. J Med Microbiol. 2009; 58(9): 1227–1230. PubMed Abstract \| Publisher Full Text\n\nMadden H, Van Andel T, Miller J, et al.: Vegetation associations and relative abundance of rodents on St. Eustatius, Caribbean Netherlands. Glob. Ecol. Conserv. 2019; 20: e00743. Publisher Full Text\n\nMarcos Tucunduva de Faria MSC, Athanazio DA, McBridea AJA, et al.: Carriage of Leptospira interrogans among domestic rats from an urban setting highly endemic for leptospirosis in Brazil. Acta Trop. 2013; 31(9): 1713–1723. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMathison BA, Pritt BS: Laboratory identification of arthropod ectoparasites. Clin Microbiol Rev. 2014; 27(1): 48–67. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRistiyanto, Mulyono A, Agustina M, et al.: INDEKS KERAGAMAN EKTOPARASIT PADA TIKUS RUMAH Rattus tanezumi Temminck, 1844 dan TIKUS POLINESIA R. exulans (Peal, 1848) DI DAERAH ENZOOTIK PES LERENG GUNUNG MERAPI, JAWA TENGAH. Vektora. 2011; 1(2 Okt), 73–83. Publisher Full Text\n\nRiyanto S: The Existence of Fleas in Rodents at Plague Observation Area in Nongkojajar Pasuruan District. Jurnal Kesehatan Lingkungan. 2019; 11(3): 234. Publisher Full Text\n\nSuku Dinas Kesehatan Jakarta Barat:Leptospirosis Jakarta Barat. Jakarta Barat. 2019.\n\nSusanna D, Nova RIT: Ectoparasites in rats and shrews data related to leptospirosis in West Jakarta, Dryad, Dataset.2021. Publisher Full Text\n\nThe ICUN Red List: Rattus rattus, House Rat Errata version. 2017.\n\nTobin ME, Fall MW: Pest Control: Rodents (Vol. II).n.d..\n\nVillanueva SYAM, Saito M, Baterna RA, et al.: Leptospira-rat-human relationship in Luzon, Philippines. Microbes Infect. 2014; 16(11): 902–910. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organization: Human Leptospirosis: Guidance for Diagnosis, Surveillance, and Control. Geneva: World Health Organization; 2003.\n\nZendehfili H, Zahirnia AH, Maghsood AH, et al.: Ectoparasites of rodents captured in Hamedan, Western Iran. J Arthropod Borne Dis. 2015; 9(2): 267–273. PubMed Abstract \| Free Full Text" }	[ { "id": "84286", "date": "11 Jun 2021", "name": "Malina Osman", "expertise": [ "Reviewer Expertise Epidemiology", "biostatistics", "tropical health" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study provided information on known reservoirs which are linked to leptospirosis. Even though the areas has been said to be endemic with leptospirosis, none of the epidemiological data is presented. It has been mentioned in the abstract that data on leptospirosis was retrieved from West Jakarta Health Office, but there is no data on leptospirosis in this study.\nIn my opinion, some statistics work should be inserted to allow any determination on correlation between number of the reservoirs with the number of cases reported.\nI would suggest molecular work on determining the presence of the bacteria in the reservoirs should be done as well.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "7694", "date": "12 Jan 2022", "name": "Dewi Susanna", "role": "Author Response", "response": "Thank you for your review, and for future research some statistics and molecular work will be inserted" } ] }, { "id": "91881", "date": "06 Sep 2021", "name": "Philip Samuel Paulraj", "expertise": [ "Reviewer Expertise Medical Entomology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article is a good paper and approved for indexing. This publication has good epidemiological significance correlating the presence of ectoparasites found on rats with the prevalence of leptospirosis.\nA similar study of mine conducted in scrub typhus endemic areas in India showed higher numbers of ectoparasites belonging to mites, fleas, and ticks. Since this study showed very few ectoparasites only belonging to fleas, an extensive study should be planned to be undertaken in these areas to collect/record more ectoparasites. This study can be taken up as a pilot study for future in-depth studies (P. Philip Samuel, R. Govindarajan, R. Krishnamoorthi, and V. Rajamannar A study on ectoparasites with special reference to chigger mites on rodents/shrews in scrub typhus endemic areas of Kerala, India Entomon, 45: 4; 20201).\nYou could also mention this reference of mine under ectoparasites identification: “Philip Samuel P., Govindarajan R., Krishnamoorthi R., Venkatesh A. (2021). A rapid protocol for clearing, staining, and mounting of Arthropoda: Trombiculidae, Pediculidae, and Pulicidae. North-western Journal of Zoology,17(1):1-5 Article No. e201104”2.\nAfter a scientific name is written in full in a publication, it is acceptable or customary to abbreviate the genus name by just using the first initial (or as recommended) and then full stop to represent the genus. These changes can be made in the entire manuscript.\nPlease mention the rat identification method.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "7693", "date": "12 Jan 2022", "name": "Dewi Susanna", "role": "Author Response", "response": "Thank you for your review and suggestion for the next research" } ] }, { "id": "99700", "date": "06 Dec 2021", "name": "Rahayu Lubis", "expertise": [ "Reviewer Expertise Epidemiology", "infectious disease", "HIV/AIDS" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis research has complete data and methods that can be carried out elsewhere. This is very beneficial for the community and it is necessary to increase early awareness of vector-borne diseases by rats and ectoparasites so that public health prevention efforts can be carried out early on, because the rat population is always present in the human population, especially in densely populated environments.\n“The type of ectoparasite found in the rats was fleas. Xenopsylla cheopis was the most common type, at 83.3% and more fleas were male, at 66.7%. The most common rat species was Rattus rattus. The ectoparasite most commonly found in them was the female flea Xenopsylla cheopis.”\nThis is an important finding because it is known that the types of ectoparasites found in rats are fleas, especially Xenopsylla cheopis, so early awareness of zoonotic diseases, especially bubonic plague, needs to be carried out in the community.\n“The technique for catching rats involved using humane live traps, while the identification of the rats and ectoparasites was done in the laboratory.”\nThis study follows ethical rules in animals.\n\nIn the method of catching rats, it is stated that the traps are set in the afternoon between 15.00 and 17.00 WIB, and the rats are collected in the morning between 07.00 and 09.00. Within 14 hours, if the rat dies and is surrounded by ants because of the smell, will it still be examined as a research sample?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "7692", "date": "12 Jan 2022", "name": "Dewi Susanna", "role": "Author Response", "response": "Thank you for your review. In this research, the rat dies will not be examined as a research sample." } ] } ]	1	https://f1000research.com/articles/10-335
https://f1000research.com/articles/10-334/v1	30 Apr 21	{ "type": "Opinion Article", "title": "Highlights from the 2016-2020 NEUBIAS training schools for Bioimage Analysts: a success story and key asset for analysts and life scientists", "authors": [ "Gabriel G. Martins", "Fabrice P. Cordelières", "Julien Colombelli", "Rocco D’Antuono", "Ofra Golani", "Romain Guiet", "Robert Haase", "Anna H. Klemm", "Marion Louveaux", "Perrine Paul-Gilloteaux", "Jean-Yves Tinevez", "Kota Miura", "Julien Colombelli", "Rocco D’Antuono", "Ofra Golani", "Romain Guiet", "Robert Haase", "Anna H. Klemm", "Marion Louveaux", "Perrine Paul-Gilloteaux", "Jean-Yves Tinevez" ], "abstract": "NEUBIAS, the European Network of Bioimage Analysts, was created in 2016 with the goal of improving the communication and the knowledge transfer among the various stakeholders involved in the acquisition, processing and analysis of biological image data, and to promote the establishment and recognition of the profession of Bioimage Analyst. One of the most successful initiatives of the NEUBIAS programme was its series of 15 training schools, which trained over 400 new Bioimage Analysts, coming from over 40 countries. Here we outline the rationale behind the innovative three-level program of the schools, the curriculum, the trainer recruitment and turnover strategy, the outcomes for the community and the career path of analysts, including some success stories. We discuss the future of the materials created during this programme and some of the new initiatives emanating from the community of NEUBIAS-trained analysts, such as the NEUBIAS Academy. Overall, we elaborate on how this training programme played a key role in collectively leveraging Bioimaging and Life Science research by bringing the latest innovations into structured, frequent and intensive training activities, and on why we believe this should become a model to further develop in Life Sciences.", "keywords": [ "NEUBIAS", "training schools", "bioimage analysis", "analyst", "workshop", "training materials", "Analyst school" ], "content": "Introduction\n\nThe NEUBIAS Training Schools (the “TSs” hereafter) have been developed as a series of 15 training events around Bioimage Analysis (BIA). These events took place between 2016-2020 over eight different venues throughout Europe (Figure 1) and brought together four communities of experts: life-scientists, microscopists, software tool developers and bioimage analysts with the aim to train the researchers and the prospective trainers, and to promote the exchange of knowledge and experience between the four communities of experts. The TSs were highly successful and well praised by the over 400 trainees, and by the recruited community of over 100 invited organisers, trainers and speakers. The series led to the creation of numerous training contents on bioimage analysis from basic to advanced topics, which are archived in repositories and are being made accessible as “open access” via the “BioImage informatics index” tool (http://biii.eu), also developed by the NEUBIAS community. The TS venues also led to the growth and strengthening of NEUBIAS as a professional network composed of bioimage analysts, software developers, instrumentalists and life scientists, with over 250 active members and over 5000 listed contacts. The interaction of the different professionals has enriched the bioimaging community hence fostering different disciplines to converge towards a common framework for BIA challenges and their research and technological needs. Here we review the conceptual design of the TSs content, how NEUBIAS tailored the level of the courses to the different needs of the aforementioned communities, and how the training programme has organically evolved over the years to cope with the tremendous pace at which BIA is developing. In the light of the TSs outcomes, the success stories and their immediate benefits for research, we discuss how instrumental international cooperation has been to support the Life Science community and the pressing need to develop a professional training programme in Bioimage Analysis in Europe, and beyond, so as to leverage local, regional, national or individual training initiatives that have substantially increased over the years of activity of the NEUBIAS network.\n\nPhotos of participants: From left to right, top to bottom: TS1 for facility staff in Barcelona, Sept. 2016 (25 trainees); TS2 for early career researchers and TS3 for bioimage analysts in Oeiras Feb. 2017 (25+25 trainees + trainers); TS4 for early career researchers & TS5 for facility staff in Gothenburg, Sept. 2017 (25+25 trainees). TS6 for early career researchers and TS7 for analysts in Szeged, Jan. 2018 (23+35 trainees); TS8 for early career researchers & TS9 for facility staff in Edinburgh, Sept. 2018 (20+20 trainees); TS10 for early career researchers & TS11 for analysts in Luxembourg, Feb. 2018 (29+39 trainees); TS12 for early career researchers & TS13 for facility staff in Porto, Oct. 2019 (28+32 participants); and finally TS14 for early career researchers & TS15 for analysts in Bordeaux, Feb. 2020 (25+35 trainees). Map chart contains the locations of the different venues, the origin of the participants and a bar chart with the distribution of applicants based on their main expertise. See also Table 1 for more information about the schools, and online at http://eubias.org/NEUBIAS/training-schools/ for more information about the programme and venues. Authorization was obtained from the participants to capture and publicize group photos or photos taken during the event.\n\n\nCentral concept(s) of NEUBIAS TSs\n\nBioimage analysis is an emerging research field, which has progressed alongside the fast-paced development of bioimaging techniques of the last couple of decades (Meijering 2007; Schneider et al. 2012; Meijering et al. 2016; Miura & Tosi 2016; Peng et al. 2012). However, the incorporation of new BIA techniques has lagged behind, leading to a bottleneck in the progress of image-based research, for which there are several reasons. First, most life-scientists who work with bioimage data lack the formal training on BIA; although imaging and bioimage data recording are now common practice in the life-sciences, training with image data analysis is still practically nonexistent in most life-sciences courses. Secondly, there is often a communication barrier between BIA algorithm developers and most end-users (so far, these have been two separate communities), which limits the assimilation of novel techniques and leads to gaps in BIA. Compared to the conventional “image analysis” in Computer Science, which aims is to mimic the human visual perception and interpretation, i.e. Artificial Intelligence, BIA aims at the objective and quantitative measurement of biological phenomena avoiding human perception bias (Miura & Tosi 2016). As visual recognition per se in most cases does not yield quantitative data, a new type of training that reaches beyond just the usage of software packages is required. Thirdly, modern scripting languages are necessary not only for automating, but also for properly and reproducibly documenting image data processing and quantitative measurements. For these reasons, more than just training about coding, the NEUBIAS TSs were designed to focus on training researchers about the “Art of Bioimage Analysis”, its scientific concepts and how to properly build and document effective workflows, using representative hands-on exercises.\n\nThe TSs were initially designed to address user requests following a 2015 survey among 2000 scientists in 32 countries worldwide (Miura, 2021). The vast majority of early career researchers and imaging facility staff did not feel well prepared to extract quantitative data from bioimages; almost 60% reported BIA as the most difficult step in image-based research, with almost 80% considering it essential or very important. Of these, nearly half reported not having access to professional support in BIA, and most importantly, an estimated 60% of image data collected is not analysed, presumably because researchers lack the proper tools and skills for processing and analysis. Support and courses were identified as the most demanded resources for (68%) researchers who rely on BIA.\n\nThe TSs followed the pioneering steps of the successful “Bioimage Analysis Courses”, organized by NEUBIAS founders and seed of its creation, in Heidelberg (2013-2017)1 as EMBL Master Courses, and in Barcelona (2013)2 together with the 1st European Bioimage Analysis Symposium (EuBIAS), building on what was then already a community-driven effort. The uniqueness of these courses consisted in the training aimed at the quantification of biological processes, more than simply tutoring the usage of image analysis software packages and plugins. Through the financial support from H2020 COST Action CA15124 from 2016, NEUBIAS was able to expand the reach and attendees-throughput of these training efforts, and prepare a new programme for TSs targeting three main audiences and perceived levels of proficiency: the i) early career researchers, who need a solid and practical understanding of BIA concepts and techniques to carry on their biological research, and an overview of the existing tools and of the limits of ethics in BIA; the ii) imaging facilities staff, whose daily tasks involve supporting researchers in BIA and who require solid skills at designing and scripting effective and reproducible workflows; and the iii) bioimage analysts who need insights into advanced utilization and scripting of BIA resources, as well as the structured designing of novel workflows. More information about the concepts, content and training cohort of the NEUBIAS TSs can be found online3,4.\n\nThe schools included sessions explaining the theoretical concepts behind the BIA techniques, a detailed explanation of a workflow exemplifying an application of the techniques, and fully documented practical exercises (with presentation slides, image datasets and exercise solutions/code). There were also guest-speaker plenary talks and ample moments for social interaction when participants (sometimes over 90) assembled at meals and coffee-breaks. Schools always concluded with “work-on-your-own-data” sessions with the support of trainers, analysts and developers, to provide the experience of a truly interdisciplinary environment. The “Analyst” schools had a somewhat different format, more workshop-style, and will be discussed in a subsequent section.\n\nWe believe the TSs were highly successful, having accepted 415 trainees out of over 900 applications from 43 different countries (see also Table 1). The rate of satisfaction averaged at 93%, and 10% of the trainees were accepted to progress through the different levels of the training programme. The seven “early career” schools trained 175 researchers (mainly life-scientists and instrumentalists), and despite being the most frequently organized they only reached 37% of the applicants, showing there is still a large community in need for training in BIA (see Figure 1 and Table 1 for applicant profiles and school statistics). The later-established online NEUBIAS Academy (explained below) was founded with this demand in mind. The four “facility staff” and four “bioimage analyst” schools, trained a total of 102 and 134 professionals, with acceptance rates of 46% and 62%, respectively. Most staff applicants considered themselves instrumentalists or analysts, and the analyst school applicants were either analysts or developers. By making this crucial effort to training professionals in imaging and analysis, instead of addressing primarily the massive demand from early career life-scientists, NEUBIAS sought to amplify the reach of the TSs, hoping that those professionals would be empowered by the training and more likely to have the time, the means and the motivation to train others locally - to guarantee this we often recruited trainees to train in subsequent schools. TSs organizers collected and catalogued numerous materials for reusing in future schools and workshops, contributing to the disseminating efforts. An excerpt of the topics and tools addressed in the schools is shown in Table 2. These include an impressive assortment of over 10 different open source toolboxes plus numerous plugins, tools and documented workflows.\n\nThese, and many others, are catalogued in Biii.eu with links to access the training materials from public repositories such as Zenodo and Github.\n\n\nChoosing topics, trainers and trainees\n\nThe topics were selected by the organizers based on perceived needs of life-scientists, the trends on publications and results of TS evaluation surveys. The TS promoted the use of open-source tools and workflows, not just to promote FAIR principles but also to guarantee easier access and applicability for all participants. The programme privileged the exploring of different tools, including learning on the advantages of one toolbox vs others; for example alternatives to visualization and rendering of 3D datasets, which is still limited in the popular ImageJ/FIJI toolbox, but better in Icy (de Chaumont et al. 2012) or Drishti (Limaye 2012); or the handling of pyramidal-multi-megapixel image data easily done with QuPath (Bankhead 2017); or preparing pipelines for recursively analysing thousands of images using CellProfiler (Carpenter et al. 2006), just to name a few. For those less familiar with scripting languages, TSs also showcased visual programming in KNIME (Berthold et al. 2008) or Icy. The schools also included sessions where trainees learned how to integrate different toolboxes to tackle complex workflows which spanned the capabilities of one particular toolbox (for example, how to integrate ImageJ/FIJI with QuPath or Imaris or Icy).\n\nDuring the unrolling of the programme there were shifts in the relative perceived importance of topics (seen also in the applications). For example, MATLAB became less requested and BIA with Python libraries and tools became a common request, which coincided with a significant increase in publication of machine/deep learning applications to BIA (Meijering 2020). Handling of “big-data” appeared also on increasing demand, and both topics became prominent in TS after 2017-2018. The choice of topics was sometimes influenced also by the coincidence with the NEUBIAS Symposia and the availability of high-profile speakers. The schools also counted with the input from the NEUBIAS taggers, a community of BIA enthusiasts who gathered during the TS events to catalogue information about software and workflows and develop online tools to help bioimage analysts, which then fed into the TSs programme.\n\nApplicants provided information that was used to match with an “ideal” profile outlined for each level of the TSs, as presented in Table 1. A committee of five evaluators read all applications anonymously and voted for acceptance based on the adequacy and preparedness of each candidate. This committee was typically composed of organizers and trainers of the current and previous events which helped maintain consistency in the selection process, and helped organizers tailor the priority topics and content of materials.\n\nThe NEUBIAS schools typically occurred in IT rooms, or in rooms prepared to accommodate for laptops (typically in the Analyst schools). Often, we paired trainees with different levels of experience to help, and all materials were prepared, tested and distributed upfront, to maintain a smoother training pace. A booklet was prepared for each school, and distributed in PDF format, with the names of participants and the synopsis of the sessions. The organizers communicated to the trainees what was the level of experience required, and sent reading materials, laptop preparation information and exercises in preparation for the schools.\n\nPresentations often included two projections, one for the basic workflow description and a 2nd used by the main presenter to demonstrate “live” for demonstrations. A team of 5-10 “helper” trainers provided constant support, with a trainer-to-trainee ratio as high as 1:2 (see Table 1). Part of the helpers’ team was actually composed of recruited “trainers-in-training”. Some TSs experimented also with parallel sessions (taking advantage of the rich pool of trainers) which allowed trainees to choose topics/techniques more in line with their research needs.\n\nAfter each school, all participants were invited to respond to a survey to provide feedback, on aspects such as duration, balance between theory and practice, preference for the tools and languages presented and methods used, other topics that should have been addressed and those that should be reduced. Each topic or session was also evaluated individually, so improvements could be made in subsequent editions. A report for each school was prepared and passed over to the next organizers of the event to try to improve and tailor the schools to the expectations of the previous trainees.\n\n\nThe “Analyst” school - a novel concept of training in Bioimage Analysis\n\nBioimage analysis is an emerging field and there was clearly a lack of formal and dedicated training for “Bioimage Analysts”. However, as the field is evolving rapidly, those who are working already as experts in their institutes do require continuous training for utilizing novel algorithms, concepts, techniques, and tools. In addition, lateral communication among those experts needs to be promoted in the school-format to strengthen their own community. To design such a training school, the highly diverse educational backgrounds of Bioimage Analysts, ranging widely among almost all fields of the life sciences, hindered us from simply implementing an \"advanced course\" because each expert is differently advanced in various specific directions. For this reason, we made a simple assumption that the minimal common denominator of Bioimage Analysts is the ability to code proficiently in at least one of major programming languages used in bioimage analysis (for example, ImageJ macro, R, Java, Python, MATLAB, C, C++). This requirement also arises counterintuitively from the high quality of current BIA software, open-source or commercial. For many well established analysis workflows there are implementations in software that can be used readily by biologists. These tools are often accompanied by useful documentation and a good user-interface, and several new tools of this desirable quality emerge every year. We reasoned that the core added value of a bioimage analyst would be the ability to address analysis tasks for which an end-user accessible tool does not exist yet. Their work then involves combining image analysis components into new workflows, which requires at least some expertise in programming (Miura et al. 2020).\n\nThis core task of analysts - creating new analysis pipelines - also shaped the following four concepts of the Bioimage Analysts School:\n\n(1) API Beating, where we invited developers of interesting implementations of image processing/analysis algorithms (components) and asked for a fast-track navigation into the Application Programming Interface (API) and the practical hands-on to API’s entry points.\n\n(2) Workflow Deconstruction, where we invited the authors of a paper with an interesting bioimage analysis workflow and asked them to explain how they solved their biological questions with their image analysis strategy. School participants examined the details of the solution, practiced code modification/migration trials with different components, refactoring, and also providing critical evaluations, which were all based on group coding (Louveaux & Verger, 2021).\n\n(3) New algorithms, where we invited the developer of the state-of-art bioimage analysis components and asked for an in-depth lecture.\n\n(4) Benchmarking, where participants were asked to benchmark currently existing implementations.\n\nIn the last Analyst school (2020), an additional 5th concept was introduced: a module for utilizing the latest statistical tool sets, such as the Tidy Data Handling (Bercowsky Rama, 2021) and the post-P value statistics (Gómez-de-Mariscal, 2019). With the recent refinement of high-content imaging technologies (e.g. High Throughput Screening), statistical analysis of rich multidimensional image data has increasingly become an important aspect of analysis workflows, and still requires to be further developed and integrated so as to become a solid part of the Bioimage Analysts training concepts. As a by-product, the Analyst TSs were leveraged also to help aspiring analysts identify with the job and give them a sense of community and profession. Overall, the Analyst TSs pioneered a unique place for the training of BIA experts. Addressing these five concepts in a three and a half day event was often a challenge, and one that is likely to be addressed in the organization of future and potentially longer NEUBIAS Analyst schools.\n\nEvery year, all contents of the Analyst courses were freshly prepared based on the initial motivation to “train bioimage analysts with the latest knowledge and techniques”. For this reason, the Analyst schools often accepted some applicants more than once, which was not possible in the other schools, where trainees could only - and where in fact, encouraged to - progress to a more advanced level of the programme. This, and the fact that the Analyst schools were more “workshop style” and therefore requiring less trainers (in the Analyst schools trainees often were presenter themselves), allowed for a gradual increase in the number of accepted applicants per school (39 at the top). This was a focused effort to train analysts to boost the dissemination of NEUBIAS TS concept, and to establish a significant pool of professionals and an influential community of bioimage analysts.\n\n\nThe training schools’ materials\n\nThe prioritized topics were identified for each school in preparatory meetings with the NEUBIAS working group that oversaw the TSs planning and invited the scientific organizers, who then took the previous programme and reports, suggested adaptations or improvements and invited more trainers. To promote continuity with the concepts and spirit of the TSs, some trainers, trainees and organizers from previous schools were invited. Efforts were made to create continuity between sessions, and to explore the possibility of featuring workflows that required a diversity of tools. Materials were stored in a cloud storage accessible by all those involved in the organization and training, and periodic meetings were done to discuss progress and consistency of the training materials being prepared.\n\nWith the collaboration of the trainers and speakers, NEUBIAS developed a collection of TS materials, which includes over 50 unique training sessions, fully documented with i) presentation slides ii) practical exercises with example data and iii) exercise solutions with instructions and code (“catch-up”). Currently, these materials are stored on an internal platform and are being catalogued and made accessible via the BioImage Informatics Index webtool5, part of the recommended interoperability resources of ELIXIR6 developed by NEUBIAS and whose content is maintained by the community as a crowd-sourced platform. These materials were openly shared among all participants during the TSs, who have reported to use them in their research and in local training efforts afterwards. Anyone interested in accessing and re-using these materials is encouraged to search in the Biii webtool, where NEUBIAS will keep the catalogue centralized and the most up-to-date links to the final repositories of these materials (see Data availability). There was an effort to try to create a consistent format and style for the presentation of the materials, including the slide formats, whenever possible. The organizers also experimented with video recording of some training sessions, when it was possible to fulfil GDPR compliance. Learning from this effort, “NEUBIAS Academy” was created in 2020 to offer recorded, yet compact, training modules for the community (more below).\n\nSome of the TSs content was further refined and prepared in the form of book chapters included in the recently edited Bioimage Data Analysis Workflows books, with the first edition7 freely available since 2019, and a second edition expected in 2021, while further publications based on the TSs training materials are envisioned to appear in the NEUBIAS F1000Research Gateway8.\n\n\nCommunity building, follow-up and future perspectives\n\nThe NEUBIAS TSs, beyond the primary take-home training for selected participants, were highly regarded as a venue for getting to know “who is who” and what is “state-of-the-art” in BIA. They were also strategically organized, when possible, together with the NEUBIAS Symposia that most TSs trainees attended, and where they were further exposed to the latest innovations in techniques and networking opportunities. Overall, the level of trainer support during TSs was highly praised and quite unusual for this type of training, even though some students commented that the pace was at times fast or overwhelming, owing to the diversity of topics addressed (Table 2). The organizers tried to balance the importance of exposing trainees to multiple tools and techniques, and of demonstrating how to explore the many existing software frameworks to tackle BIA problems and construct workflows. This is arguably another key aspect of NEUBIAS TSs, in contrast to other training initiatives with BIA modules but more oriented to specific biology-driven applications (e.g. 3D developmental imaging courses9) or imaging-technology (e.g. Lightsheet EMBO courses10). It was often explained that there is no “single tool for all tasks”, and that a well-prepared analyst should be ready to employ different tools.\n\nThe recruitment of trainees to serve as trainers in ensuing TSs is a significant outcome and has led also to several local training efforts that followed using the concept, methods and materials of the NEUBIAS TSs. Several trainees have reported becoming professional staff members or analysts themselves, and on the strong impact the attendance of the TS had on their career decision and CV (see, for example Box 1 containing “success stories”). Similarly to the recent establishment of national interest groups for Bioimage analysis by NEUBIAS stakeholders in their own country (e.g. IAFIG11 in UK, Swissbias12), new training initiatives derived from the NEUBIAS TSs community, such as a recently awarded EMBO course, focused on “advanced Methods in Bioimage Analysis13. Robert Haase’s Youtube content14, with over 1.000 subscribers, also benefited from NEUBIAS TS content. Other courses that followed on the footsteps of NEUBIAS TSs include the “Introduction for Image Analysis for life science” courses in Gothenburg15, or the “advanced ImageJ Macro” course by the Max Planck Postdoc Net16. Another initiative was recently funded under the auspice of EOSC Life (2nd Training Open call17) for the organisation of “BioImage Analysis Defragmentation” Training Schools, which aim to continue training the new generation of bioimage analysts on workflow-based BIA with a focus on integrating methods for cloud-based and High-Performance computing applied to life sciences. This initiative also shows the current efforts of integration of the BIA community with other life science communities and infrastructures, and paves the way to better integration and reusability of bioimage analysis results within wider analysis workflows that include technologies other than imaging.\n\nAnna Klemm. While being a staff scientist at the Ludwig-Maximilians University (LMU) in Munich (Germany), she participated at TS1 and TS3 as a trainee. She then engaged as trainer in TS4, TS10, TS13, TS14 and as scientific organizer of TS8. She became a professional full-time bioimage analyst at the SciLifeLab BioImage Informatics Facility, Uppsala (Sweden) in 2018. Since January 2021 she has been Head of the facility.\n\nMarion Louveaux. She attended TS7 as a trainee and became a trainer in TS10, 13 & 15 and co-organizer in TS10 & TS15. She is now an application specialist for the bioimage analysis software Icy at the Bioimage Analysis Unit, Institut Pasteur, Paris (France). “The Analyst school was a great opportunity to discover and connect to a network of professionals sharing the same interest”, she says, “and to consolidate an informal training in bioimage analysis. Later on, it also became a great opportunity to train others and co-organise training school events. This professional experience is still going on, with the NEUBIAS Academy, and the upcoming EOSC training school, and was probably key in securing a job with the Icy software team.”\n\nNuno P. Martins. Former imaging facility staff member at the Gulbenkian Institute of Science in Oeiras (Portugal), he attended TS1 & 7 as a trainee, and later became a trainer in TS2, and co-organizer of TS4. He is now pursuing a PhD in BIA at Max Planck Institute for Cell Biology and Genetics in Dresden MPI-CBG (Germany). He believes: “The TSs were a gateway to a community of interesting people with a lot of experience in BIA, advice and ideas on how to better do it”, and were instrumental for both the previous job at the facility and for the current PhD research.\n\nRobert Haase. Formerly a core-facility staff scientist, and later postdoc at MPI-CBG in Dresden (Germany), he has become an analyst and tool developer (Haase et al. 2020). He joined NEUBIAS TSs as a trainee in TS3, joined TS8 as trainer and acted as scientific organizer of TS13. Since 2021, he is a group leader of “Bioimage Analysis Technology Development” at the DFG Cluster of Excellence “Physics of Life” at the Technische Universität Dresden (Germany). His employment strategy follows the emerging career path promoted by NEUBIAS: In his group, life scientists with interest in BIA evolve towards becoming BIA experts, with career perspectives towards becoming BIA tool developers, lecturers or core-facility leaders.\n\nBut perhaps the most prominent follow-up of the NEUBIAS Training schools was the start of the “NEUBIAS Academy”18. This new initiative stemmed from former trainers and trainees of the NEUBIAS TSs, with the aim of developing a community-driven framework for training in BIA. It has contributed to expand enormously the reach of the NEUBIAS training programme, especially during the COVID-19 pandemic, by starting to hold regular live webinars in 2020, and producing accessible online content. To date, NEUBIAS Academy has produced 28 webinars which attracted over 14,000 registrations from all continents. Webinars are recorded and publicly available on the NEUBIAS YouTube Channel19 (>54,000 views, as of March 2021). While continuing to hold such webinars with increased focus onto thematic series (e.g. “Big Data Analysis Series”, “Correlative and Multimodal Analysis”20), the NEUBIAS community envisions to resume in-person training as satellite events of the NEUBIAS conference, which has become a reference meeting point for the bioimage analysis community. The recent enthusiasm and community engagement witnessed with the NEUBIAS Academy webinars show that the interest and demand for training in BIA, as well as the community, are still growing. We believe this interest has been expanded by the success stories of the TSs, which were carried worldwide by the hundreds of participants. The fact that the TS reached such a widespread community (see Figure 1) certainly helped raise awareness among life science researchers that this training is fundamental, but more importantly, that it is available and supported by a large and active community, ready to engage and to supply solutions.\n\nWhile local and national initiatives are slowly coming into place in several countries in Europe or overseas21, the consolidation of Bioimage Analysis as a key research field able to leverage bioimaging resources and life science research will require in the coming years a sustained international coordination and new funding mechanisms to continue building similar training capabilities, at high levels of quality and quantity to match the current needs. A similar effort developed recently is the I2K event “From Images to Knowledge with ImageJ & Friends” (2018; 202022), with a similar community-building ethos, and a large audience of BIA enthusiasts and developers. Given the increase in demand for BIA training, and the trend for remote training in times of social distancing and climate change, we envision future TSs in mixed format to explore the availability of online resources and webinars, with concurrent “in-person” trainings organized locally at different locations worldwide. The coordinated organization of these events would include shared topics, speakers/presenters, training materials, concepts, training practice and discussions following the NEUBIAS TS style. This promotes international cooperation, standardized methods and recognized levels of expertise, broader dissemination of training efforts, empowers local initiatives to contribute and benefit from the resources of a more global community, and will build on the spirit of a community-gathering that has become a hallmark, and highly praised, characteristic of NEUBIAS events. Ultimately, we have raised the interest and the level of awareness that hopefully will contribute to the inclusion of formal training in BIA in life-sciences curricula, at least at a basic level. The NEUBIAS community is hereby open to participate in efforts to create new higher-education curricula and teaching contents on BIA.\n\n\nData availability\n\nNEUBIAS TS materials are catalogued and made accessible via the BioImage Informatics Index webtool (http://biii.eu), where NEUBIAS will keep the catalog centralized and the most up-to-date links to the final repositories of the materials (Zenodo and GitHub, for those that are complete and ready to be shared publicly via a CC-BY 4.0 Creative Commons License).", "appendix": "Acknowledgements\n\nBesides the funding bodies, the organizers of the NEUBIAS training schools would like to thank all school organizers and trainers, and their host institutions for supporting them, including the Carpenter and the Eliceiri Labs for supporting trainers from the US, and also all contributors of training materials and data - their names and contributions can be found online at the NEUBIAS website. We particularly acknowledge all in kind and financial contributions of hosting institutions, entities and local organizers of the eight TS events, as well as their administrative, technical and catering staff: Universitat Pompeu Fabra in Barcelona; Fundação Calouste Gulbenkian in Lisbon and Instituto Gulbenkian de Ciência and Bioinformatics Training (Pedro Fernandes) in Oeiras; Center for Cellular Imaging and Sahlgrenska Academy at University of Gothenburg, The University of Szeged, Institute of Informatics and the Biological Research Center of the Hungarian Academy of Sciences in Szeged; MRC Centre for Regenerative Medicine, Univ. of Edinburgh, and the Univ. of Dundee; Luxembourg Center for Systems Biomedicine, Life Science Research Unit (LSRU), and Luxembourg Centre for Systems Biomedicine (LCSB), Univ. of Luxembourg; Institute for Molecular and Cell Biology IBMC at i3S and INESC TEC - Institute for Systems and Computer Engineering, Technology and Science, and the Porto Convention & Visitors Bureau in Porto; Bordeaux Imaging Center and Institute for Interdisciplinary Neuroscience IINS at University of Bordeaux, and the Bordeaux Métropole. We also thank the Institute for Research in Biomedicine (IRB Barcelona) for financially coordinating NEUBIAS Training Schools under the COST funding programme.\n\nThis publication was supported by COST Action NEUBIAS (CA15124), funded by COST (European Cooperation in Science and Technology).\n\n\nReferences\n\nBankhead P, et al.: QuPath: Open source software for digital pathology image analysis. Sci Rep. 2017. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBercowsky Rama A: Python: Data handling, analysis and plotting. In: Bioimage Data Analysis Workflows - advanced Components and Methods. Cham: Springer International Publishing; 2021. in press.\n\nBerthold MR, et al.: KNIME: The Konstanz Information Miner. In: Preisach C, Burkhardt H, Schmidt-Thieme L, Decker R. (eds) Data Analysis, Machine Learning and Applications. Studies in Classification, Data Analysis, and Knowledge Organization. Berlin, Heidelberg: Springer; 2008. 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Learning Materials in Biosciences. Cham: Springer International Publishing; 2020. Publisher Full Text\n\nMiura K, Tosi S: Introduction: What is Bioimage Analysis? In: Bioimage Data Analysis, edited by Kota Miura. Weinheim: Wiley-VCH; 2016; 1–3. 978-3-527-80094-0.\n\nMiura K, Paul-Gilloteaux P, Tosi S, et al.: Workflows and Components of Bioimage Analysis. In: Bioimage Data Analysis Workflows, edited by Kota Miura and Nataša Sladoje, 1–7. Learning Materials in Biosciences. Cham: Springer International Publishing; 2020. Publisher Full Text\n\nPeng H, Bateman A, Valencia A, et al.: Bioimage informatics: a new category in Bioinformatics. Bioinformatics. 2012; 28, 1057–1057. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchindelin J, et al.: Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012; 9: 676–682. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchneider C, Rasband W, Eliceiri K: NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012; 9: 671–675. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\n\nFootnotes\n\n1 http://wiki.cmci.info/shared/bias2013 and other links thereafter.\n\n2 http://eubias2013.irbbarcelona.org/bias-2-course.\n\n3 http://neubias.org/NEUBIAS/venue/neubias/training-wg2/.\n\n4 http://neubias.org/NEUBIAS/training-schools/.\n\n5 http://www.biii.eu.\n\n6 https://elixir-europe.org/platforms/interoperability/rirs.\n\n7 https://link.springer.com/book/10.1007/978-3-030-22386-1.\n\n8 https://f1000research.com/NEUBIAS.\n\n9 https://meetings.embo.org/event/18-developmental-imaging.\n\n10 https://meetings.embo.org/event/18-lsm.\n\n11 Image Analysis Focused Interest Group of the Royal Microscopy Society: https://iafig-rms.org/about.\n\n12 SwissBIAS: The Swiss BioImage Analysts’s Society. https://swissbias.ch/.\n\n13 https://www.embl.org/about/info/course-and-conference-office/events/bia21-01/.\n\n14 https://www.youtube.com/playlist?list=PL5ESQNfM5lc7SAMstEu082ivW4BDMvd0U.\n\n15 https://www.gu.se/en/core-facilities/graduate-course-introduction-in-image-analysis-for-life-science.\n\n16 https://www.bioimagingnet.mpg.de/events/26607/6105.\n\n17 https://www.eosc-life.eu/services/training/.\n\n18 https://neubiasacademy.org/.\n\n19 https://www.youtube.com/neubias.\n\n20 In partnership with COMULIS, COST Action CA17121, https://www.comulis.eu/.\n\n21 The Center for Open Bioimage Analysis: https://openbioimageanalysis.org/.\n\n22 https://www.janelia.org/you-janelia/conferences/from-images-to-knowledge-with-imagej-friends." }	[ { "id": "84282", "date": "07 May 2021", "name": "Thorsten Wohland", "expertise": [ "Reviewer Expertise Biophysical Fluorescence" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMartins et al. report on the outcome of NEUBIAS, a COST Action to create a network of European bioimage analysts. NEUBIAS has over the years run training schools at different levels from beginners to professionals and helped create a community of bioimage analysts well beyond the borders of Europe that connects between researchers in and practitioners of bioimage analysis. NEUBIAS has created very effective training schools that not only train interested participants but “trains the trainers”, which led to several local efforts to provide training courses in different countries using the same concepts that NEUBIAS used. In addition to training and networking, NEUBIAS has been involved in the creation and publication of bioimage analysis protocols and workflows and has helped train bioimage tool developers and facility managers. This report explains the core concepts of NEUBIAS and how it achieved its goals. This can function as a blue print for other communities with similar needs for networking and the dissemination of specialist knowledge in a diverse community to create well sought after experts in new upcoming fields.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] }, { "id": "84283", "date": "18 Jun 2021", "name": "Kevin Eliceiri", "expertise": [ "Reviewer Expertise image informatics", "image analysis", "imaging", "cell biology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present an important aspect of the NEUBIAS, the European Network of Bioimage Analysts, the training schools. These schools were a central educational tool of NEUBIAS and were done as 15 events around biomaging analysis. During the four year term of the program, over 400 trainees attended, and involved over 100 invited organizers, trainers and speakers. The paper does a nice job of outlining the rationale behind the innovative three-level program of the schools, the curriculum, the trainer recruitment and turnover strategy, the outcomes for the community and the career path of analysts, including some success stories. There are important lessons to be shared from this program that could guide future such events. As such the enthusiasm for this article is very high.\nThe only missing element in this reviewer's opinion is some sort of discussion in surveying of how the attendees were directly impacted. It is stated the attendees well received this program and some success stories are given but no direct metrics. For example, it was stated that some survey of student knowledge going into the program (pre-training) was assessed but there was no mention of student knowledge gained from the school (post training). This is a lost opportunity. If available it would be good to have a discussion and/or table to see in what areas attendee's knowledge of image analysis improved. If this data is not available some discussion of future need for concept evaluation would be helpful\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-334
https://f1000research.com/articles/10-333/v1	30 Apr 21	{ "type": "Software Tool Article", "title": "ImageM: a user-friendly interface for the processing of multi-dimensional images with Matlab", "authors": [ "David Legland", "Marie-Françoise Devaux", "Marie-Françoise Devaux" ], "abstract": "Modern imaging devices provide a wealth of data often organized as images with many dimensions, such as 2D/3D, time and channel. Matlab is an efficient software solution for image processing, but it lacks many features facilitating the interactive interpretation of image data, such as a user-friendly image visualization, or the management of image meta-data (e.g. spatial calibration), thus limiting its application to bio-image analysis. The ImageM application proposes an integrated user interface that facilitates the processing and the analysis of multi-dimensional images within the Matlab environment. It provides a user-friendly visualization of multi-dimensional images, a collection of image processing algorithms and methods for analysis of images, the management of spatial calibration, and facilities for the analysis of multi-variate images. ImageM can also be run on the open source alternative software to Matlab, Octave. ImageM is freely distributed on GitHub: https://github.com/mattools/ImageM.", "keywords": [ "image processing", "matlab", "multivariate image analysis", "segmentation", "interactive segmentation" ], "content": "Introduction\n\nModern imaging devices provide a wealth of data often organized as images with many dimensions, for instance 2D/3D, time and channel. This leads to new challenges in the exploration, visualization and processing of multi-dimensional data. In particular, multi-spectral images require the application of methods at the intersection of spectroscopy and image processing.\n\nMatlab (The Mathworks, Natick, MA), is an efficient software solution for image and signal processing that provides native support for multi-dimensional arrays, a large number of image processing methods, and a great facility for adding custom developments. However, it lacks many features for facilitating the interactive interpretation of image data, such as a user-friendly visualization of multidimensional images, or the management of image meta-data (e.g. spatial calibration), thus limiting its application to bio-image analysis.\n\nThe ImageJ/Fiji software (Schneider et al., 2012) is an alternative that provides an intuitive graphical user interface for image exploration, and many image processing functions that take into account spatial or channel calibration. The basic functionalities can by extended by a large collection of plugins. The integration of custom developments is possible via macros or plugins, but this often requires advanced programming skills.\n\nThe ImageM software aims at providing a user-friendly interface for the interactive exploration, processing and analysis of multi-dimensional imaging data within the Matlab environment, while taking into account meta-data and facilitating the integration with custom algorithm developments.\n\n\nMethods\n\nImageM strongly relies on Matlab’s Object-Oriented Programming. The core feature is an “Image” class that handles arrays up to five dimensions, corresponding to three spatial dimensions, the channels, and the time. It also encapsulates image meta-data such as spatial calibration, image name, or channel names.\n\nThe user interface relies on various image viewers adapted to the type (grayscale, color, label, multivariate) and the dimensionality of the image (Figure 1a). Interactive tools allow for quick image exploration, for example histograms, line profiles and 3D visualization. The architecture was kept modular to facilitate the inclusion of new features.\n\nOriginal 3D image is visualized via adapted viewer (a). Image processing operators are applied (b), using contextual dialogs (c). Segmentation results are represented by label images (d,g). Analysis of the image results in table frame (f). Menu actions are transcripted into runnable Matlab commands (e). Original image courtesy of K. Belcram (Moukhtar et al., 2019).\n\nTo have the whole functionalities, Matlab (v2020a) is required, as well as the Image Processing and the Statistics toolboxes. The Gui Layout Toolbox (Sampson, 2021) is required to benefit from flexible image viewers. ImageM runs on a typical workstation. The application is launched by typing “ImageM” from the command line.\n\nMenus provide quick access to common image processing operators, organized in a way to make them easily discoverable by a non-expert user. When necessary, intuitive dialogs allow for setting up parameters. Preview of the results facilitates the choice of relevant settings. Region of interest may be defined interactively to perform interactive measurements.\n\nTo facilitate usage during scripting and integration with custom developments, images can be easily imported from and exported to the workspace. Applying an operator results in a log entry that can be integrated into a script for quickly generating a workflow from the succession of operations.\n\nImageM integrates a large family of image processing operators (linear filtering, mathematical morphology, segmentation). Intuitive dialogs allow tuning of parameters and preview of the result, facilitating the choice of relevant settings (Figure 1c). The type of the result image is automatically inferred to provide the most adequate visualization (Figure 1d and e).\n\nSegmentation algorithms result in binary or label images that can be used for quantitative analysis of regions. The spatial calibration of images is considered when available. Features can be visualized as geometric overlays on the image or exported to data tables (Figure 1f).\n\nMulti-channel images or microscopy images contain pixels represented of several values. Multivariate image analysis provides a convenient formalism to analyze such images by taking account both the spatial and the spectral dimensions (Geladi & Grahn, 2006). ImageM can convert multi-variate images into data tables, allowing their exploration through multi-variate analysis tools such as Principal Component Analysis or k-means clustering (Figure 2). Resulting tables can be easily back-converted to images to facilitate the visual interpretation.\n\nLeft panel: original multivariate image (from Devaux, 2008). Middle panel: application of K-means clustering on principal components. Right panel: color representation of k-means classes.\n\nImageM can also run within the open-source platform, Octave, after some modification of the code (see Extended data (Legland & Devaux, 2021)).\n\nWe present here a use case describing image segmentation and region analysis of the 3D image presented in Figure 1. The Extended data (Legland & Devaux, 2021) contains the sample files, and more detailed use cases.\n\n• First, run the application by typing “ImageM” from Matlab command line.\n\n• In the menu, select “File->Open Image …” and choose the file “arabidopsis-embryo.tif”. This opens an image viewer for the 3D image.\n\n• Reduce the noise in the image, by selecting “Process->Gaussian Filter …”. This opens a dialog.\n\n• Choose value 1 for each of the “sigma” values, and click “OK”. The filtered image appears in a new viewer.\n\n• Perform 3D segmentation of the cells. Select menu entry “Process->Extended Min Watershed …”. Choose a basin dynamic equal to 6, the “C6” connectivity, and result type as “basins”. This results in a new 3D label image in a new viewer, where each label is displayed with a different color.\n\n• Some regions do not correspond to cells. They can be removed manually. Select the menu “Process->Replace Value(s) …” and fill in with “1, 2, 28, 29” (values can be identified by moving the mouse cursor and inspecting the status bar).\n\n• Morphometric features can be computed from 3D label images, by selecting the menu “Analyze->Analyze Regions …”. After choosing the features and clicking “OK”, the selected features appear in a new data table frame that can be saved as a text file or exported to the workspace.\n\n• The 3D representation of the Figure 1g can be obtained by selecting the menu entry “View -> Show 3D isosurface”, choosing a “smoothing radius” value equal to 1 and checking the options “Reverse Z-axis” and “Rotate Ox”.\n\n\nSummary\n\nThe ImageM application proposes a convenient user interface for the visualization and exploration of multi-dimensional images within Matlab. It also provides basic support for regions of interests, the management of data tables. Its modular architecture should facilitate the future incorporation of new functionalities. The current work focuses on a better management of regions of interest, and on the inclusion of user plugins.\n\n\nSoftware availability\n\nPackaged application available for direction download from: https://www.mathworks.com/matlabcentral/fileexchange/45847-imagem\n\nSource code available from: https://github.com/mattools/ImageM\n\nArchived source code as at time of publication: http://doi.org/10.5281/zenodo.4674326 (Legland, 2021).\n\nLicense: BSD 2-Clause “Simplified” License\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nZenodo: Extended data for the manuscript “ImageM: a user-friendly interface for the processing of multi-dimensional images with Matlab”, http://doi.org/10.5281/zenodo.4705240 (Legland & Devaux, 2021).\n\nThis project contains the following extended data:\n\n- User manual\n\n- Octave compatibility notice\n\n- Two detailed use cases\n\n- Images used in use cases\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nThis publication was supported by COST Action NEUBIAS (CA15124), funded by COST (European Cooperation in Science and Technology).\n\n\nReferences\n\nDevaux M-F: UV and visible fluorescence images of maize stem: macroscopy and confocal microscopy. [Data set]. Published 2018 via Perscido-Grenoble-Alpes. Reference Source\n\nGeladi P, Grahn HF: Multivariate Image Analysis. In: Encyclopedia of Analytical Chemistry (eds Meyers RA, Meyers RA). 2006.\n\nLegland D: mattools/ImageM: ImageM v1.3.2.1 (Version v1.3.2.1). Zenodo. 2021, April 9. Publisher Full Text\n\nLegland D, Devaux M: Extended data for the manuscript “ImageM: a user-friendly interface for the processing of multi-dimensional images with Matlab” (Version 1.0). Zenodo. 2021, April 20. Publisher Full Text\n\nMoukhtar J, Trubuil A, Belcram K, et al.: Cell geometry determines symmetric and asymmetric division plane selection in Arabidopsis early embryos. PLOS Computational Biology. 2019; 15(2): 1–27. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSampson D: GUI Layout Toolbox. MATLAB Central File Exchange. Retrieved April 13, 2021. 2021. Reference Source\n\nSchneider CA, Rasband WS, Eliceiri KW: NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012; 9(671-675): 2012. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "89355", "date": "23 Jul 2021", "name": "John Anthony Jose", "expertise": [ "Reviewer Expertise Computer Vision", "Image Processing" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors are able to clearly explain the problem and contribution of the paper. However, the method of how they validated their contribution is quite limited. Specifically, they mentioned that the problem they would like to solve is that the existing approaches on visualizing and exploring multi-dimensional images lacks features in interactive interpretation. Concretely, it is about the lack of user-friendly visualization and management of image meta-data. However, their results didn’t provide any validation that their contribution is has better user-friendly approaches compared to the existing one.\nThe authors can improve on their article by concretely discussing on how their results was able to directly address the problem that they are trying to solve. Additionally, it will be helpful if they provide comparisons with existing software for multi-dimensional image analysis.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [] }, { "id": "92537", "date": "09 Sep 2021", "name": "Curtis Rueden", "expertise": [ "Reviewer Expertise Software engineering", "software architecture", "image visualization", "image processing", "image analysis." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nImageM is a MATLAB-based application, backed by a modular collection of MATLAB libraries, for performing image processing and analysis on multidimensional scientific images with the common dimensions of X, Y, Z, channel, and time. Modeled closely after ImageJ, ImageM integrates many powerful routines into a central main window, making them more easily accessible, especially to those less versed in writing scripts.\nThe use cases do a nice job of quickly showcasing some of the great things you can do with ImageM. However, the article's introduction and rationale needs work, and the program needs to be better documented and tested. Nonetheless, this software is certainly worth advertising to the scientific community.\n> Is the rationale for developing the new software tool clearly explained?\nPartly. I have two problems with the rationale as written:\nThe claim that ImageM addresses novel visualization challenges:\n> Modern imaging devices provide a wealth of data often organized as images with many dimensions, for instance 2D/3D, time and channel. This leads to new challenges in the exploration, visualization and processing of multi-dimensional data. In particular, multi-spectral images require the application of methods at the intersection of spectroscopy and image processing.\nChallenges in visualization of 3D, time and channel are not \"new\". ImageJ's core gained 5D support in 2007 with version 1.39l, and there were many earlier programs with >=5D support as well (e.g. BioImageXD, VisBio, ImageJ via the Image5D plugins, OME Server).\nHighlighting multi-spectral as a challenge makes sense, but it is hardly the only dimension-related challenge of scientific imaging. Multiple angles? Fields of view? Fluorescence lifetime?\nImageM is limited to 3D+time+channel. Therefore, it does not facilitate handling of any \"new challenges\" in dimensionality beyond this paradigm.\nRather than argue that ImageM addresses new challenges in scientific imaging, the article would be better served explaining that ImageM builds on the wealth of functionality available in MATLAB, enabling more seamless integration of other MATLAB-based tools with its 5D image paradigm. The two use cases, especially use case 2, illustrate this fact well.\n\nThe lack of discussion of the technical merit of a MATLAB-based implementation of an ImageJ-like program:\nWhile this is a short article, it would be ideal—if sufficient space—to mention ImageJ's MATLAB integrations ImageJ-MATLAB (https://imagej.net/scripting/matlab) and MIJ/Miji (https://imagej.net/plugins/miji), and how ImageM compares to them. This is an important part of the justification for essentially reimplementing ImageJ in MATLAB: what are the downsides of using the existing ImageJ within MATLAB via its built-in JVM? How does ImageM overcome those downsides?\nI do believe there is technical merit to ImageM. But the article needs to do a clearer job explaining the benefits.\n> it lacks many features for facilitating the interactive interpretation of image data, such as a user-friendly visualization of multidimensional images or the management of image meta-data (e.g. spatial calibration), thus limiting its application to bio-image analysis.\nI am not well versed in the ecosystem of MATLAB add-ons, so I cannot evaluate whether this statement is really true, but I am skeptical. Consider rewording to frame things more positively, saying that ImageM provides user-friendly capabilities that build upon, enhance, and complement MATLAB's existing image processing offerings.\n> The integration of custom developments is possible via macros or plugins, but this often requires advanced programming skills.\nI disagree about MATLAB programming being easier than ImageJ macro programming. Both are designed to be as accessible as possible, while still being powerful. Instead of arguing that extending ImageJ is too hard, why not instead argue that extending ImageM via MATLAB code enables scripters to easily harness MATLAB's power, flexibility, and extensive suite of toolboxes and add-ons?\n> Are sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others?\nYes. Use case 1 was fully reproducible for me. Use case 2 was also fully reproducible, but:\nThere appears to be some randomness to the K-Means clustering output. My results, while functionally identical, differed from the screenshot regarding the cluster numbering.\n\nWhile following the steps, the following error is emitted to the command window:\nError using convhull Error computing the convex hull. The points may be collinear.\nError in Table/scatterGroup (line 204)\n\ninds2\n\n= convhull(xdata(inds), ydata(inds));\nError in imagem.actions.table.process.TableKMeans/run (line 88)\n\nscatterGroup(tab(:,1), tab(:,2), k);\nError in imagem.gui.FrameMenuBuilder>@(src,evt)action.run(obj.Frame) (line 475)\n\n'Callback', @(src, evt) action.run(obj.Frame));\n\nError while evaluating Menu Callback.\n\nFolding the k-means score table back to an 800x800 image does not emit a line of script for programmatic reproduction.\nAll of that said, the use case of K-means clustering to segment tissue types is very cool!\nRelatedly, ImageM is hard to install:\nI could not get the ImageM Octave fork working in Octave. I recommend removing the claim that ImageM works with Octave. (Which is a shame, because a major problem with MATLAB is that it is not open source.)\n\nI could not get ImageM working in MATLAB Online. This isn't too surprising, but may want to specifically acknowledge that it doesn't work in the online app.\n\nI could not get ImageM working in a local MATLAB R2021a after installing it from the Add-On Manager, even after also installing the add-on dependencies MatImage, geom2d, geom3d, and MatStats. Is there really no dependency management system for MATLAB add-ons? If not, that is a huge downside of using MATLAB compared to Java, Python, JavaScript, Ruby, or other popular language ecosystem.\n\nI was finally able to get ImageM to work by opening the ImageM-1.3.2.1-full.mlappinstall bundle downloaded from the GitHub releases page. It added a working ImageM icon to MATLAB's APPS tab. The documentation—especially the GitHub repository's README.md and ImageM page on MATLAB Central—needs to explain in much more detail how to install this software.\n\nI continued to receive startup errors about the MATLAB path due to backslashes in the path strings. Based on the errors I received trying to run it with backslashes in paths, it seems like ImageM is only tested on Windows. I was testing ImageM on Ubuntu Linux 20.04 LTS.\nMore generally, ImageM's documentation is lacking:\nWhy is the manual only a PDF? Why not online as HTML or Markdown?\n\nThere is no way to quick-launch commands from the menu. A command finder or search bar would be very helpful, especially as people start developing ImageM extensions that add to the menu structure.\n\nThe Help menu should offer links to important resources, especially the online docs.\nFinally, a couple of miscellaneous comments:\nMathworks named the program MATLAB in all caps (\"MATLAB\"), not title case (\"Matlab\").\n\nFrom the ImageM manual:\n\n> An element of an image is usually associated to a square region centered on its coordinates.\n\n> Its bounds are therefore ±0.5 around each dimension.\nA pixel is not a little square. http://alvyray.com/Memos/CG/Microsoft/6_pixel.pdf\nAs mentioned above, I had a hard time getting ImageM installed. Here are the issues I filed on GitHub to give some technical detail:\nMATLAB Online – https://github.com/mattools/ImageM/issues/1 Octave – https://github.com/mattools/ImageM/issues/2 MATLAB R2021a – https://github.com/mattools/ImageM/issues/3\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-333
https://f1000research.com/articles/9-1368/v1	25 Nov 20	{ "type": "Systematic Review", "title": "Rapid systematic review of systematic reviews: what befriending, social support and low intensity psychosocial interventions, delivered remotely, are effective in reducing social isolation and loneliness among older adults? How do they work?", "authors": [ "Elisabeth Boulton", "Dylan Kneale", "Claire Stansfield", "Paul Heron", "Katy Sutcliffe", "Brenda Hayanga", "Alex Hall", "Peter Bower", "Dympna Casey", "Dawn Craig", "Simon Gilbody", "Barbara Hanratty", "Dean McMillan", "James Thomas", "Chris Todd", "Claire Stansfield", "Paul Heron", "Katy Sutcliffe", "Brenda Hayanga", "Alex Hall", "Peter Bower", "Dympna Casey", "Dawn Craig", "Simon Gilbody", "Barbara Hanratty", "Dean McMillan", "James Thomas", "Chris Todd" ], "abstract": "Background: During the 2020 COVID-19 pandemic, millions of older adults are advised to avoid contact with those outside their household. ‘Social distancing’ has highlighted the need to minimise loneliness and isolation through the provision of remotely delivered befriending, social support and low intensity psychosocial interventions. We wanted to know what interventions are effective and how they work to help inform decisions about different approaches. Methods: We followed a systematic ‘review of reviews’ approach and included systematic reviews focussed on the effectiveness or implementation of remote interventions to reduce levels of social isolation or loneliness in adults aged 50+. Searches of 11 databases were undertaken during April 2020 and eligible reviews were critically appraised using AMSTAR2. Narrative synthesis was used at a review and study level to develop a typology of intervention types and their effectiveness. Intervention Component Analysis (ICA) and Qualitative Comparative Analysis (QCA) were used at a study level to explore the characteristics of successful interventions. Results: We synthesised evidence from five systematic reviews and 18 primary studies. Remote befriending, social support and low intensity psychosocial interventions took the form of: (i) supported video-communication; (ii) online discussion groups and forums; (iii) telephone befriending; (iv) social networking sites; and (v) multi-tool interventions. The majority of studies utilised the first two approaches, and were generally regarded positively by older adults, although with mixed quantitative evidence around effectiveness. Focussing on processes and mechanisms, using ICA and QCA, we found that the interventions that were most effective in improving social support: (i) enabled participants to speak freely and to form close relationships; (ii) ensured participants have shared experiences/characteristics; (iii) included some form of pastoral guidance. Conclusions: The findings highlight a set of intervention processes that should be incorporated into interventions, although they do not lead us to recommend particular modes of remote support.", "keywords": [ "social isolation", "loneliness", "remote interventions", "review of reviews" ], "content": "Introduction\n\nDuring the 2020 coronavirus (COVID-19) crisis, millions of older adults over 70 years old have been advised to be particularly stringent about social distancing, and to avoid contact with those outside their household1. Older adults are more likely to have long-term illness or disability, to live alone and to be widowed, all of which are risk factors for loneliness2. Social isolation and loneliness adversely affect quality of life, wellbeing and mental health, and are associated with physical ill health and mortality3. Social distancing and restrictions on face-to-face contact increase the risk of social isolation and loneliness. The requirement for older adults to restrict their activities during the COVID-19 pandemic puts a spotlight on the need to understand how to minimise the impact of loneliness and isolation using remotely-delivered approaches.\n\nIn the voluntary and community sector, many existing services are shifting to providing remote support, often via the telephone. In England, the call during March 2020 for NHS Volunteer Responders included roles to make ‘regular phone calls to check on people isolating at home’4. Fulfilment of such roles requires that:\n\n(i) the programmes and interventions staffed by these volunteers are effective and have minimal adverse consequences for older people; and\n\n(ii) the volunteers making phone calls and providing other forms of support are adequately trained and supported to fulfil these roles, with training based on evidence of how the intervention should be delivered and the key proesses that generate successful interventions.\n\nThis review focusses on interventions that seek to ameliorate loneliness or social isolation, or both. We conceptualise loneliness as an emotional response by individuals when there is a ‘deficit between their desired and actual quality and quantity of social engagement and relationships5, p64’. We define social isolation as ‘having minimal quantity and quality of structural and functional support’ which can involve having social networks of low density that are not maintained through frequent engagement6, p15. Structural support reflects the number and diversity of social contacts and social roles; functional support reflects the meaningful functions that these social relationships play. Both loneliness and social isolation are conceptually distinct from living alone, the latter having limited utility as a proxy for either social isolation or loneliness7.\n\nA number of evidence reviews have highlighted the diverse range of interventions to alleviate loneliness amongst older adults in a variety of settings8,9. In the main, these have been face-to-face interventions, either in groups or between individuals. During the height of the COVID-19 pandemic these interventions were of limited utility as lockdown regulations in many countries confined the vast majority of the population to their homes, except for essential outings. In this period all opportunities for face-to-face social contact outside the home were curtailed, and visiting friends and family for social contact prohibited. Even as these regulations were eased social distancing has restricted opportunities for social interaction, by restricting face-to-face connections and physical contact. During this period there has been considerable growth in the use of remote communication tools including telephones, videoconferencing, or other internet ‘chat’ facilities.\n\nThis rapid review examines evidence on whether befriending, social support, and low intensity psychosocial interventions delivered remotely can reduce social isolation or loneliness among older adults. Specifically, the aims are to:\n\n(i) Identify existing systematic reviews on befriending, social support, and low intensity psychosocial interventions delivered remotely for older adults.\n\n(ii) Synthesise review-level findings on the nature and effectiveness of these interventions.\n\n(iii) Generate new understandings on how interventions work and which core components and processes are associated with successful interventions.\n\n(iv) Map the review-level and study-level evidence to better understand evidence gaps.\n\nThis paper is an abridged summary of a full report, available elsewhere, containing further details10. The rapid review was conducted in a short timescale (four weeks for the main body of work), and adopted a review of reviews approach to meet these timescales.\n\n\nMethods\n\nWe followed a systematic ‘review of reviews’ methodology to synthesise evidence from related (but differing) interventions for social isolation and loneliness, to help inform decisions about different approaches11.\n\nThe review adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) checklist for the reporting of systematic reviews12. A protocol was agreed before data extraction and published on the EPPI-Centre website.\n\nSearches of 11 bibliographic databases and online resources across the fields of health, social care, psychology and social science were carried out on 23rd-24th April 2020. We searched: Applied Social Sciences Index and Abstracts (ASSIA)(Proquest), Emerging Sources Citation Index (Web of Science), Database of Promoting Health Effectiveness Reviews (DoPHER), Epistemonikos, Medline (OVID), NHS Evidence, PsycInfo (OVID), Social Policy and Practice (OVID), Social Sciences Citation Index (Web of Science), Social Systems Evidence and Sociological Abstracts (Proquest).\n\nThe search terms reflected four concepts that needed to be present in each of the study citations:\n\n1) Population: older and middle-aged populations aged 50+ years.\n\n2) Interventions that enable remote delivery: technology, remote communication, telephone, helplines, self-help, bibliotherapy.\n\n3) Outcomes: loneliness, social isolation (or close proxy measures e.g. social contact).\n\n4) Study design: systematic reviews.\n\nAn example search history for Medline is presented in the full report10 and as Extended data69.\n\nInclusion and exclusion criteria were also based on the Population, Intervention, Comparator, Outcome and Study Design (PICOS) framework:\n\nPopulation: We included reviews on ‘older’ adults age 50+ (see protocol for further details13). Participants could be located in a variety of settings in the community or residential care, although reviews of interventions delivered to older adults in hospital settings were excluded. Studies included older people who were socially isolated, lonely, or who were otherwise at risk of loneliness or isolation.\n\nWhile we identified all reviews on older people, we only synthesised evidence from reviews focussed on diverse populations of older people. We did not synthesise evidence from reviews focussed exclusively on particular groups of older people, specifically older caregivers (see 10), although interventions including caregivers are well represented in the evidence presented. The descision to synthesise evidence from a subset of reviews was inline with the rapid timescales of the review (see (Figure 1).\n\nIntervention: Included reviews examined interventions that sought to reduce levels of social isolation or loneliness, through strengthening individuals’ social contacts and social relationships (e.g. befriending and social support), or through low intensity psychosocial interventions (e.g. internet-delivered CBT - iCBT), using remote methods and technologies. Interventions were delivered on a one-to-one basis (e.g. befriending), or as remote group-based interventions (e.g. discussion groups). We did not include interventions that examined the use of social robots, pets or virtual pets, or reviews solely focused on the use of technology for educational or training purposes.\n\nComparator/control: We included reviews that included studies with most forms of control group (randomised and non-randomised) and those without a control group (pre-post designs). Reviews on the implementation of interventions, including qualitative evidence syntheses were also included.\n\nOutcomes: Included reviews measured social isolation or loneliness as a primary outcome. Based on previous reviews, we expected various measures of loneliness and social isolation to be reported, and we included both bepsoke measures and proxies, such as reports of social connectedness.\n\nStudy design: We defined systematic reviews as those that met at least four of the following criteria14:\n\n1. Were inclusion/exclusion criteria reported?\n\n2. Was the search adequate?\n\n3. Were the included studies synthesised?\n\n4. Was the quality of the included studies assessed?\n\n5. Were sufficient details about the individual included studies presented?\n\nWe did not include any other reviews of reviews, but used these to identify additional systematic reviews. We included unpublished manuscripts. We did not place any restrictions on date of publication, although only reviews in English were selected.\n\nWe exported search records to EPPI-Reviewer web15 and de-duplicated the records. Title and abstract screening was undertaken independently by three reviewers (DK, EB, PH) following joint screening of 204 citations (10%) to ensure consistency. For records included for full-text screening, each record was examined in duplicate, and reviewers met online to reconcile any differences. Reasons for exclusion are reported in Figure 1.\n\nSystematic reviews in this area often include a mix of eligible and ineligible interventions. In line with previous overviews16, and in addition to the criteria outlined above, systematic reviews were included if they:\n\n(i) contained only or a majority of interventions within scope; or\n\n(ii) contained separate evidence tables, or defined sections of evidence tables, presenting evidence on interventions within scope; or\n\n(iii) contained separate synthesis sections presenting evidence on interventions within scope.\n\nInterventions in scope were befriending, social support, and low intensity psychosocial interventions, delivered remotely, to reduce social isolation and loneliness among older adults. We did not include reviews where only a single study within the review met our criteria. Individual studies reported within systematic reviews were identified as relevant, using the same inclusion criteria as above (albeit applied at the study, not review level) and after agreement of two reviewers.\n\nData were extracted by two reviewers and any differences agreed in online reconciliation meetings. We extracted the following data from reviews:\n\n• Lead author and team;\n\n• Year of publication;\n\n• Number of primary studies included in the review;\n\n• Primary study design(s) (e.g., RCT studies, qualitative studies);\n\n• Aims of review and main topic focus; (e.g. if focussed on social isolation/loneliness);\n\n• Target population (e.g., if focussed on particular group e.g. bereaved older people);\n\n• Participant characteristics (e.g., age, gender);\n\n• Intervention approaches in primary studies (e.g., type of remote intervention);\n\n• Synthesised outcomes/key findings relating to social isolation and/or loneliness; secondary outcomes relating to implementation and adverse effects;\n\n• Quality assessment characteristics and rating.\n\nIncluded systematic reviews were critically appraised using AMSTAR-217 by two reviewers (DK/BH and EB/PH). Criteria were summed and categories of quality created based on the AMSTAR-2 assessment (low risk of bias: equivalent to high confidence in AMSTAR-2; unclear: equivalent to moderate confidence; and high risk of bias: equivalent to low or critically low confidence).\n\nDescriptive analysis of reviews and studies. We produced textual descriptions of the reviews and their findings and presented this in tabular form to develop a preliminary understanding of the evidence. The results also helped to populate an evidence map (see later synthesis).\n\nNarrative synthesis of the evidence. A narrative synthesis was conducted to examine review-level and study-level findings. The narrative synthesis focussed on the outcomes of befriending, social support, and low intensity psychosocial interventions delivered remotely. Building on the descriptive analysis, we followed guidance outlined elsewhere18.\n\nIntervention Component Analysis and Qualitative Comparative Analysis. We drew on two complementary synthesis methods – Intervention Component Analysis (ICA) and Qualitative Comparative Analysis (QCA) – and applied these to primary studies contained within the reviews that presented quantitative results, to understand how interventions ‘worked’. The first approach, ICA19, is an inductive approach developed in response to the poor reporting of intervention processes. It involves (a) inductively coding the nature of intervention features (i.e. components) and (b) using trialists’ informally-reported experiences of implementing the intervention (i.e. information usually located in introduction and discussion sections of trial reports, which is usually not incorporated into analysis)19. This information is then used in conducting the QCA.\n\nThe second approach, QCA, is applied to numeric data and is based on set-theory20. QCA is employed as a solution to the challenge of analysing data containing a small number of studies (known as cases in QCA terminology), each with an extensive array of factors that may trigger a given outcome21. This ‘small N-many variables’ challenge is similar to that faced by systematic reviewers, and Thomas and colleagues provide one of the first examples where QCA was utilised within a systematic review to understand configurations of intervention components that were aligned with ‘successful’ interventions22. Studies were eligible for QCA if they reported quantitative findings (see Results). We identified studies as belonging to both ‘condition sets’ (i.e. belonging to a distinct set of studies distinguished by the presence or absence of different characteristics or processes) and ‘outcome sets’ (i.e. belonging to a group of studies differentiated by whether they are considered most effective or least effective). Ultimately, we were interested in establishing which condition sets ‘overlapped’ with successful outcome sets. The goal of QCA is to identify the simplest expression of characteristics/processes that lead to effective interventions; to find the simplest expression we drew on Boolean minimisation. We followed standards of good practice that have been laid out elsewhere in conducting the QCA23. Further explanation of the approach is provided alongside the results.\n\n\nResults\n\nThe literature searches identified 2,715 citations. After duplicates were removed, 2,057 citations were screened at title and abstract level, identifying 75 possible studies for inclusion. Full texts were obtained for all 75 records, with nine potential reviews identified and five included for synthesisi (see Figure 1). Not all of the primary studies within these five reviews met our inclusion criteria (see Methods) and from the 112 primary studies included across the five reviews, we identified 18 studies as eligible for synthesis.\n\nReview populations. The reviews covered a range of populations, using different definitions and age thresholds for ‘older adults’, with a combined age range of 50–95. The settings were not always clearly stated, but were primarily older adults’ own homes, nursing homes, or supported living facilities, in North America, Europe and Taiwan. Whilst some reviews contained studies focused on the general older adult population, others included studies of people with multiple chronic conditions, specific conditions (such as Alzheimer’s Disease, or breast cancer), or in a particular geographical area.\n\nReview study designs. RCTs, quasi-experimental cohort studies, survey studies, and qualitative (semi-structured interviews and focus groups) were all represented. Three of the five reviews conducted quality appraisals on the included studies25–27, one evaluated only the effectiveness of the technologies within the studies, not the quality of the study itself28, and one did not report any quality appraisal29ii.\n\nThe reviews contained studies reporting interventions using various technologies to deliver remote befriending, social support or low intensity psychosocial interventions including those in scope (e.g. video-communication and telephone befriending) and those out of scope (e.g. computer training and internet training). There was a range of different outcome measures within the reviews, although all contained some measure of loneliness or social isolation.\n\nRisk of bias assessment of included reviews. All of the reviews were deemed to be of low or critically low quality (displayed as having a high risk of bias in Table 2). Although all had reasonably clearly defined PICO components and had conducted reasonably comprehensive search strategies, the majority had failed to prepare a protocol, and many failed to justify the choice of study selection. This latter concern was particularly problematic where authors had included studies of various designs.\n\nPrimary study characteristics. Befriending, social support and low intensity psychosocial interventions reported in the 18 primary studies fell into five categories reflecting modes of delivery:\n\nSupported videoconferencing to alleviate loneliness34–38.\n\nTelephone befriending to reduce social isolation39,40.\n\nOnline discussion groups/forums to reduce social isolation and/or loneliness, or to improve/maintain social connectedness40–47.\n\nSupported use of social networking sites for mitigating social isolation and loneliness48.\n\nMulti-tool interventions (PC, training, messaging, chat groups) to reduce loneliness and/or social isolation, or increase social connectedness44,47,49,50.\n\nFurther primary study characteristics, including their populations, details of implementation, methodological details including how the outcome was measured, and outcomes as found in Table 1.\n\n+=low risk of bias (equivalent to high confidence); ?=moderate or unclear (equivalent to moderate confidence); - = high risk of bias (equivalent to low or critically low confidence); Note chart shows only those items relevant to all included reviews.\n\nSupported videoconferencing to alleviate loneliness. Four reviews included a total of three qualitative studies, three quantitative studies, and one mixed-methods study, on supported video-communication interventions. Five studies involved supporting older adults to communicate with family members35–38,51, with the other two reporting on the videoconferencing element of the ACTION service in Sweden and Norway34,46. The qualitative evidence suggests the interventions were generally regarded positively by older adults, with increased feelings of connection with their family members35. The quantitative evidence showed some evidence of decreases in feelings of loneliness and increases in social support scores. Two quantitative studies37,38 found reduced feelings of loneliness at one week, three months and 12 months, although this achieved statistical significance in only one studyiii37. Torp et al.’s46 mixed-methods pilot cohort study, employing questionnaires and focus groups, also found that the video phone was important for building and maintaining relationships. It is important to note that all interventions included ongoing support to use the technology. See Table 1 for contextual details of reviews and studies on videoconferencing.\n\nTelephone befriending to reduce social isolation. Two reviews included a total of one qualitative and one quantitative study reporting on forms of telephone contact, one of which was a study of telephone befriending. Cattan et al.39 reported on the Call in Time intervention, with qualitative findings from 40 participants. Telephone calls were made to older adults by volunteers, with a project co-ordinator managing the process. Findings included reduced feelings of social isolation, loneliness, depression and anxiety; improved state of mind, contentment with life, confidence level, and physical health (less pain). This study built on an earlier evaluation report that presented data used for the QCA52; this evaluation report was not directly included in any of the reviews, but quantitative data presented within this report suggested that participants had lower wellbeing and social support after the intervention, albeit with a number of caveats.\n\nThe only other included study to incorporate telephone contact was Gustafson et al.40, from the Morris et al.27 review, where one element of the intervention was to match up participants with peer advocates, who engaged in weekly phone calls. This was not a telephone befriending service, as the peer advocate had a different role to that of a befriender. Findings showed that, of those who used a peer advocate 77.3% felt somewhat or very much connected with their peer advocate, and 81.6% felt that the peer advocate helped them cope (somewhat or very much so) with their breast cancer. Perceived social support increased significantly over the four months, but the intervention included more elements than just telephone support (computer and internet training, discussion group, ‘ask an expert’ service and written guidelines).\n\nOnline discussion groups/forums to reduce social isolation and/or loneliness, or to improve/maintain social connectedness. Two reviews contained eight quantitative studies and one mixed-methods (questionnaires and focus groups) on online discussion groups and forums. The studies included synchronous and asynchronous communication: real-time chat discussions, instant peer-messaging, email contact with professionals, and discussion boards. Interventions were designed to support women with chronic illness or breast cancer40,44,47,50 ; people with diabetes or heart transplant recipients41–43; and caregivers of people with dementia or stroke survivors45,46. The qualitative evidence suggested that discussion groups helped older adults to build social networks and friendships and to feel more familiar with people through regular connections45,46. The quantitative evidence showed mixed results with regard to loneliness and social isolation. The majority of studies showed increases in social support, but only two showed reductions in loneliness, with four studies not measuring loneliness at all. The asynchronous chat room ‘Koffee Klatch’ in Hill et al.’s44 primary study provided a forum for women with chronic illnesses to share their feelings, concerns, life experiences and provide support to each other over 22 weeks, resulting in significant improvement in social support, but not in loneliness, compared to the control group. The Sharing Circle in Weinert et al.47 provided the same opportunities, with the addition of discussion of self-study units and internet-based health information. This study saw statistically significant improvements in loneliness, but not in social support, compared to the control group. See Table 1 for details of reviews and studies on online discussion groups/forums.\n\nSupported use of social networking sites for mitigating social isolation and loneliness. Two reviews included the same study on social networking sites48. The authors of this qualitative study found that the utilisation of a bespoke social networking site had the potential to reduce loneliness in older people, as there were positive impacts on temporal loneliness (especially in the evening) and on connectedness. Review authors suggested that older adults were more interested in a smaller number of strong relationships mediated through the internet, than they were in a larger network with weak ties. They report that perceived value could have been an issue for older adults, which may have been more obvious through supported social networking service interventions such as that reported by Ballentyne et al.48.\n\nMulti-tool interventions (PC, training, internet use, messaging, chat groups) to reduce loneliness and/or social isolation, or increase social connectedness. Three reviews included a total of four quantitative studies on multi-tool interventions. van der Heide et al.49 report on the Care TV package for people receiving home care in The Netherlands. This video and voice network allowed clients to communicate round-the-clock with a nurse practitioner. They received a ‘Good Morning/Goodnight’ call and could use the video facility to call family members. Average feelings of loneliness decreased substantially, with social and emotional loneliness showing pronounced decreases. The three other studies reported on web-based discussion groups in the Women-to-Women programme, with mixed results regarding levels of loneliness and social support. Weinert et al.47 reported on an RCT of a web-based discussion groups, with a peer-led online support group and self-study units supported by an Advance Practice Nurse. Improvements were found in loneliness, but there was no significant difference in social support between the intervention and comparison groups, following the 11-week intervention. Weinert et al.50 found significant increases in both loneliness and social support, compared to the control group, over the 22-week intervention. Hill et al.44, found statistically significant effect on both social support and loneliness after 22 weeks. See Table 1 for contextual details of reviews and studies on multi-tool interventions.\n\nQCA and ICA were undertaken to help us further identify the processes and mechanisms that were common across the interventions described in Table 1 and the narrative synthesis. To undertake QCA, we first conducted ICA to understand the nature of the interventions. We inductively coded the nature of intervention features (i.e. components) and used trialists’ informally reported experiences of implementing the interventions (e.g. author reflections reported in introduction and/or discussion sections) to understand the importance and underlying mechanisms of particular features20.\n\nTheory selection and setting up the QCA. A fundamental element of QCA is the selection of an appropriate theory to base the analysis on, and to help identify suitable evidence to extract as part of the ICA. To understand which processes might be important to incorporate in interventions – regardless of specific mode of delivery (i.e. videoconferencing or internet chat group) – we drew on Robert Weiss’s53 ‘Fund of Sociability’ theoryiv. The theory is intended to capture assumptions, content, and functions of social ties that can help to support developing social relationships. The theory specifies six characteristics of social interactions and relationships that are necessary for well-being and the avoidance of loneliness53. Table 3 outlines the six categories, their definitions and how we interpreted them in relation to the interventions in the QCA.\n\nOur QCA built on the earlier descriptive and narrative synthesis, and addressed the question: ‘Do the characteristics of social interactions and relationships stated in the fund of sociability theory explain differences between remotely delivered interventions found to be effective compared to those found to be ineffective?’ To gain familiarity with the studies and attempt to gain ‘deep case knowledge’, we started by reading and re-reading the studies.\n\nSelection of studies (cases)v for the QCA. We focussed on studies that met our criteria for the QCA including that they (a) presented quantitative results, (b) were remotely delivered, (c) focussed on older people, and (d) actively sought to strengthen social relationships or prevent/offset loneliness. From the 18 primary studies described above, 12 met these criteria.\n\nDeveloping a data table. QCA is based on set-theory with sets differentiated as belonging to a successful and unsuccessful set on the basis of their outcome. The outcome can be based on an objective measure or subjective or quality measure54, and on a single measure or a composite indicator55. The allocation of studies into a successful set and unsuccessful set can follow different strategies. Firstly, success may be defined through the observation of clinically or statistically significant change in the outcome (for example56). A second approach is for the researcher to set thresholds for determining success. A third strategy is ensure (approximately) equal representation of un/successful cases by ranking cases according to their effectiveness and allocating studies into un/successful outcome sets. A fourth strategy is to use a more qualitative approach where additional characteristics besides the outcome value are considered to ensure a representation of studies in the un/successful outcome set57. In a systematic review, stratifying studies by their characteristics to ensure a diversity in study size or study design among successful studies may ensure a more informative solution is produced.\n\nTo generate our outcome sets, and group interventions as being ‘successful’ or ‘unsuccessful’, we calculated an estimated effect size for each study. Effect sizes are used differently within QCA as opposed to meta-analysis; i.e. as a guide to allocating studies to successful (most effective) or unsuccessful (least effective) outcome sets, rather than to provide a pooled estimate of effect with precision. Most effect sizes were based on measures of social support, which we regarded as a proxy measure for social isolation. The exceptions were Schwindenhammer36 and O'Connor, et al.45 where a measure of loneliness was the only suitable outcome available. However, we did attempt to express the effect sizes in a common rubric where possible, e.g. prioritising post-test measures for studies that involved randomisation of participants or clusters (five studies), and change measures where these data were not available. For those studies with a comparator group (eight studies), effect sizes were calculated in the standard way see Thomas et al., 201758; for those studies that employed a pre- and post- evaluation design an effect size was estimated based on changes in the pre- and post- individual scores divided by the standard deviation at pre-test59,60; in some cases this involved using mean differences as proxy information and other approximations.\n\nUsing the effect size for indicative purposes, we grouped interventions into those that were ‘successful’ (studies with effect sizes over 0.5), ‘partially successful’ (studies with effect sizes between 0.2 and 0.5) and ‘not successful’ (studies with effect sizes under 0.2 or suggested negative impacts) based on thresholds suggested by Cohen61 for interpreting effect sizes. However, combining the different study designs, and particularly those with and without a comparison group, using the same approach could lead us to overstate the effectiveness of studies without a comparison group. To mitigate this possibility we also present the results of a sensitivity analysis, where we imposed an additional ‘penalty’ on studies without a control group – studies with effect sizes of 0.5 and over were deemed to provide partial evidence of success (0.66); studies with effect sizes between 0.2 and 0.5 were deemed to provide weak evidence of being ‘not successful’ studies (0.33); while studies with values lower than 0.2 were deemed to provide strong evidence of being ‘not successful’ (0). This is akin to adding in additional ‘qualitative’ information – in this case on study design – to distinguish studies as belonging to a successful and unsuccessful outcome set57. We also examined the potential impact of omitting these four studies, although this is not a preferred option given that QCA models typically need 10 or more cases as a minimum.\n\nTo create our data table, a coding scheme was developed to determine whether the conditions reflecting the fund of sociability processes were actually present in the studies (see 10). The results of this coding and the data table are presented below in Table 4.\n\nSee also notes in methodology for further explanation.\n\nTruth table. As we had a limited set of cases for the number of conditions, our analytical strategy involved first creating a ‘truth table’ based on six conditions, and then producing a reduced truth table containing four conditions and minimised solution24. A ‘truth table’ sorts cases according to the configuration of conditions they exhibit. Although we noted that both ‘availability’ and ‘control’ were conditions generally only observed in successful intervention studies, they did not appear to be as critical to outcome success as the other conditions, appearing in fewer studies. Our reduced truth table thus contained four conditions (intimacy, interaction, support and nurturance) with five of a possible 16 configurations represented (Table 5). Two configurations are observed as triggering a successful outcome; in one, supported by five studies, all four conditions are present; in the second, supported by two studies, three of four conditions are present. On the right side of the table is a column marked consistency; this indicates the strength to which studies that belong to the condition set are also a subset of the outcome set. A value of 1 indicates perfect consistency; all cases in the configuration are strong members of the condition set and the successful outcome set; and there is strong evidence that these intervention characteristics trigger successful outcomes. A value of 0 indicates perfect inconsistency and there is no evidence that these intervention characteristics trigger successful outcomes. Values in between indicate some degree of ambiguity, which was expected given that we used a “fuzzy-set coding scheme” which allowed studies to be partial members of sets (using a value of 0.85 to denote membership).\n\nBoolean minimisation and formation of a solution. We applied Boolean minimisation to obtain the simplest expression of those conditions (intervention processes) that were associated with triggering a successful intervention. We developed a complex solution based on the observed data only, and found that those interventions that ensured the following processes took place were those in the successful outcome set:\n\nINTIMACY and INTERACTION and SUPPORT\n\nWithin QCA, information from unobserved configurations (logical remainders) can be used to simplify the solution and check the quality of the solution. We incorporated these logical remainders to develop two further solutionsvi, although incorporating logical remainders in this model did not help to simplify the solution above. Our model and details of its fit are presented below (Table 6). The high consistency value for the solution suggests that when this configuration of conditions is observed in an intervention, it is generally sufficient to trigger a successful intervention (i.e. a substantial change in social support). The coverage statistic suggests that the model broadly accounts for the successful interventions observed.\n\nSensitivity and additional technical quality checks. Using an alternative measure of effect size that incorporates a ‘penalty’ reflecting the greater uncertainty around pre-post studies, we re-ran the analysis described above. The truth table (Table 7) with this alternative outcome showed one configuration of successful studies. This suggested that studies which incorporated all three processes observed earlier, as well as nurturance, were those that triggered a successful outcome (using a slightly lower consistency value of 0.825, which is still well within recommended thresholds24). Coverage was slightly lower for this solution, although the solution still provided a comprehensive explanation of why some interventions were successful (Table 8). The replication of the same three core conditions provides a degree of triangulation that our main solution identified in Table 6 provides a robust account; the inclusion of nuruturance as an additional condition below is not contradictory, but suggestive that as a condition it may help to distinguish a smaller pool of studies as successful.\n\nWe also undertook quality checks to understand whether our solution, or the assumptions we made in its derivation, could also predict unsuccessful outcomes, and found little evidence that this was a possibility. We also explored whether focussing only on the 10 studies that measured social support would change our interpretation, and again found little evidence that this would influence the model. Similarly, focussing only on studies that had a comparison group showed a similar pattern descriptivelyvii.\n\nInterpretation of the solution. The successful outcome set contained those interventions that: (i) supported participants to form ‘intimate’ relationships and express their feelings freely without self-consciousness between peers; (ii) ensured that there were shared characteristics between participants and their peers (beyond a single experience, and beyond geography alone); and (iii) included some form of pastoral care or support (e.g. light-touch oversight of a discussion forum by professionals or opportunities for participants to contact professionals for advice). This configuration explained the majority of the successful outcomes we observed.\n\nTaken together, albeit with some caveats, these characteristics can form a set of design principles for future interventions that are delivered remotely which aim to increase support available to older adults and offset the risks of social isolation and loneliness. The interventions that were not in the successful outcome set did not provide evidence that all three processes had been part of the interventions, and some indicated that processes to the contrary had taken place.\n\n\nSummary and discussion\n\nIn this rapid review of reviews, narrative synthesis showed that supported video-communication interventions are regarded positively by older adults and can have positive effects on loneliness and social support. However, the quantitative evidence remains uncertain and, although they were placed in the effective set of studies in QCA analysis, uncertainty about effectiveness is a shared conclusion in other similar reviews62. Evidence about online discussion groups and forums also demonstrated mixed results, with increases in social support, but less evidence for improvements in loneliness. Telephone befriending has not been widely researched, but qualitative evidence suggested this intervention model may be helpful in addressing loneliness and social isolation, although quantitative evidence did not show this. The evidence for social networking sites was weak. Multi-tool interventions showed decreases in loneliness, but not always increases in social support. Clearly, these interventions vary greatly, so it is difficult to isolate the effective elements. Similarly, conceptualisations of loneliness and social isolation vary, making comparisons and conclusions challenging.\n\nUsing QCA, we looked beyond specific models of intervention to explore which intervention processes are aligned with being in an effective intervention set. We have shown that the following processes are enabled in effective interventions including (i) supporting development of intimate relationships; (ii) supporting interactions through ensuring participants share experiences/characteristics; (iii) supporting participants through pastoral guidance.\n\nGaps in the evidence. Despite our extensive searches, we found only one study of telephone befriending39 included within a single systematic review. This was also the one of the few studies that made use of volunteers. There was no information provided about the training and support provided to the volunteers, as the focus was on the experience of older adults receiving the service. Similarly, we found little information about training and support provided to staff members supporting other forms of intervention. Information, communication, support, moderation and mediation was provided to older adults by research staff and health professionals (nurses, psychologists, advance nurse practitioners) within the primary studies, but there was little detail about how staff (paid and volunteer) were trained or supported to provide these. The evidence identified in the QCA finds that successful interventions are effective because they are able to ehance complex psychosocial processes and abilities, highlighting that staff may need specialist training in delivering interventions successfully. In addition, support and training is likely to be important for managing the wellbeing of those delivering the intervention. Guidelines published elsewhere suggest volunteers or staff members should receive high-quality training and regular supervision to be competent63, yet the call for NHS Volunteer Responders to make telephone calls to isolated older adults did not include any offers of training or support4.\n\nMost of the studies included in this review involved some form of new technology, with just two involving an intervention delivered through (traditional) telephone. No study examined an intervention delivered through a smartphone. Similarly, our inclusion criteria could have theoretically allowed other forms of remotely delivered interventions to be included, such as letter writing, although no such study was identified. These forms of interventions could be purposively considered in future reviews, with a recent intervention involving cross-generational letter writing suggestive of positive impacts for older and younger people alike64. There may be scope in the future for inter or cross-generational interventions that can help to provide both befriending, and technological support, while maintaining the principles outlined earlier.\n\nWe found few studies reporting on low intensity psychosocial interventions, which could be due to our focus on loneliness and/or social isolation as outcomes of interest. In the broader literature, whilst some studies have demonstrated positive impacts on depression, wellbeing and general mental health of delivering therapies through remote means65, several of these interventions may not specifically address loneliness and are not targeted at older adults66.\n\nEmpowering and supporting older adults involved in remote interventions. Overall, the results here suggest that older adults can be empowered to support each other through online discussions and forums. In the narrative synthesis we found reviews containing several studies with peer support, provided through synchronous and asynchronous messaging, chat rooms and discussion forums. This challenges the assumption that older adults must always be on the receiving end of an intervention to address social isolation and loneliness. When we moved to study-level synthesis, we also found that studies that enabled older people to feel that their contributions could improve the outcomes of others (i.e. improved levels of self-worth40–42,46,50) tended to be successful interventionsviii. As the mobilisation of thousands of volunteers takes place to support older people who are currently shielding in the COVID-19 pandemic, recognising that older people can be both providers and recipients of support simultaneously is likely to be an important principle to adhere to in the design of activities.\n\nStrengths and limitations. The strengths of this rapid systematic review of reviews include the transparent and robust approach to searches, data extraction, review quality appraisal and analysis, ensured through pre-publication of a protocol on the EPPI-Centre website. Despite the rapid nature of this review process, we have conducted the review according to systematic review methodology67. In this case, the rapid element of the review was primarily reflected in the decision to exclude reviews focussed on caregivers from the synthesis; other stages were conducted according to standard systematic reviewing practice. A further strength was the diversity of synthesis approaches conducted, including QCA and ICA.\n\nSearching for systematic reviews means that we may have missed some more recent primary studies in this area, but it ensured that our review was achievable within the four-week timeframe required for a timely response during the COVID-19 crisis. We applied the AMSTAR2 quality appraisal tool to the included reviews, although the reviews included in the synthesis were found to have a low quality rating. In addition, we did not conduct any quality assessment of the primary studies that we looked at in more detail. Some of these had been assessed by the review authors, but many had not. There were very few identifiably robust primary studies that met our inclusion criteria. Only one primary study was identified by review authors as ‘strong’, with others rated as ‘weak’ or with no quality appraisal at all. The poor, or lack of, quality rating for many of the included studies means that findings should be considered with caution. In addition, few of the studies considered potential adverse impacts of the interventions. However, this is the case for many reviews in this research area and is not unique to our rapid review.\n\nOwing to the rapid nature of this review, we focussed on reviews addressing interventions to mitigate loneliness or social isolation on the general older adult population. This meant we excluded reviews identified through the searches focussing exclusively on caregivers that may have provided additional insights. Other limitations included our treatment of primary studies in the QCA, where neither the precision of the effect size, study design, or quality were included in the model or the allocation into different outcome sets in our main model. Studies with weaker designs, and effect sizes derived from these, were treated in the same way as those with more robust designs in our main model. Although this is not uncommon in QCA practice, further synthesis could be conducted focussing on only those studies with a more robust design in future.\n\nFurther research and conclusions. Loneliness and social isolation are extremely complex phenomena6, and require a deep understanding and deliberative treatment that was beyond the remit of this rapid review. The risk of running unsuccessful interventions may be higher than many triallists appear to recognise, and a failure to ensure that the processes identified as important in effective interventions are incorporated into intervention design may have adverse impacts for older people, for example in heightening their feelings of alienation68. Our findings do not lead us to recommend one particular mode of delivering befriending, social support, or low-intensity psychosocial interventions over another (e.g. videoconferencing, telephone calls, chat rooms or forums), and all may be of benefit, but our findings do suggest that the principles highlighted from the QCA should be incorporated into the delivery of an intervention.\n\nWe were surprised by the identification of only one systematic review including a telephone befriending intervention. Given the UK Government’s interest in encouraging volunteers to make phone calls to physically isolating and shielding older adults, under the ‘stay at home’ guidance, a systematic review of telephone befriending interventions is needed, to identify evidence to inform policy in this area. A review by Sharma et al.69 suggested that a large portion of such interventions may be found in grey literature. In the current context of the COVID-19 pandemic, a number of befriending interventions are being delivered by a variety of organisations, and there is scope to incorporate learning from these in future systematic reviews in this area.\n\nAs the training and support components of the technology-mediated interventions were unreported in the reviews and studies that we synthesised, there is a need to search for these elsewhere. Evaluations of existing telephone befriending and psychosocial support services, often found in the grey literature, could act as a starting point. Additional valuable information could be obtained through contact with voluntary sector and NHS organisations delivering befriending, peer support and low-intensity psychosocial interventions. A review of these training and support components could add valuable insight for policy-makers and service providers to ensure that volunteers are well trained, empowered and supported in delivering interventions adhering to the principles outlined earlier. Although we believe all of the intervention modes in scope here have the capacity to include the processes found to lead to more successful interventions (supporting the development of intimate relationships; supporting interactions through ensuring participants share experiences/characteristics; provide pastoral guidance), a more encompassing piece of research is needed in order to identify which mode is most effective, or has the greatest potential, for changing outcomes.\n\n\nData availability\n\nAll underlying data as published on the Open Science Framework\n\nOpen Science Framework (OSF): Rapid systematic review of systematic reviews: what befriending, social support and low intensity psychosocial interventions, delivered remotely, are effective in reducing social isolation and loneliness among older adults? How do they work? https://doi.org/10.17605/OSF.IO/VS2UX13\n\nThis project contains the following underlying data:\n\n- Data and example evidence for studies included in QCA and conditions included in truth tables.docx\n\n- Data extracted on reviews and primary studies for narrative synthesis.docx\n\n- Data used for QCA.csv\n\nOpen Science Framework (OSF): Rapid systematic review of systematic reviews: what befriending, social support and low intensity psychosocial interventions, delivered remotely, are effective in reducing social isolation and loneliness among older adults? How do they work? https://doi.org/10.17605/OSF.IO/VS2UX13\n\nThis project contains the following extended data:\n\n- Search history - medline example.docx\n\n- Further details of exclusion criteria.docx\n\nOpen Science Framework (OSF): PRIMSA checklist for “Rapid systematic review of systematic reviews: what befriending, social support and low intensity psychosocial interventions, delivered remotely, are effective in reducing social isolation and loneliness among older adults? How do they work?”. https://doi.org/10.17605/OSF.IO/VS2UX13\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nThis paper represents independent research commissioned and funded by the National Institute for Health Research Policy Research Programme. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR, the Department of Health and Social Care, or its partners.\n\n\nFootnotes\n\ni Four of the reviews focussed exclusively on interventions to support caregivers: 30. Corry M, Neenan K, Brabyn S, et al. Telephone interventions, delivered by healthcare professionals, for providing education and psychosocial support for informal caregivers of adults with diagnosed illnesses. Cochrane Database of Systematic Reviews 2019(5); 31. Elvish R, Lever S-J, Johnstone J, et al. Psychological interventions for carers of people with dementia: A systematic review of quantitative and qualitative evidence. Counselling and Psychotherapy Research 2013;13(2):106-25.; 32. Hopwood J, Walker N, McDonagh L, et al. Internet-based interventions aimed at supporting family caregivers of people with dementia: systematic review. Journal of medical Internet research 2018;20(6):e216.; and 33. Lins S, Hayder‐Beichel D, Ruecker G, et al. Efficacy and experiences of telephone counselling for informal carers of people with dementia. Cochrane database of systematic reviews 2014(9). In view of the need for rapid evidence synthesis, we excluded these reviews from the synthesis, which only included the reviews focussed on the general older population (this included care givers in some instances).\n\nii Note that while Beneito-Montagut and colleagues’ study (2018) was a self-defined ‘review of the literature’, it was deemend to sufficiently meet the criteria of a systematic review for this review of reviews.\n\niii It was also unclear whether the intracluster correlation had been accounted for in calculating measures of effect.\n\niv This theory also served as the conceptual framework underpinning one of the included studies 49. Weinert C, Cudney S, Hill WG. Rural women, technology, and self-management of chronic illness. The Canadian journal of nursing research= Revue canadienne de recherche en sciences infirmieres 2008;40(3):114.\n\nv Terminology is used when conducting QCA that is distinct from other research approaches. 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Publisher Full Text" }	[ { "id": "76527", "date": "25 Jan 2021", "name": "Syed Ghulam Sarwar Shah", "expertise": [ "Reviewer Expertise Public Health", "Loneliness", "Digital technology for health", "Health inequalities", "Systematic reviews and meta-analysis", "Quantitative research", "Patients’ access to electronic health/medical records." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMany thanks for inviting me to review the manuscript entitled “Rapid systematic review of systematic reviews: what befriending, social support and low intensity psychosocial interventions, delivered remotely, are effective in reducing social isolation and loneliness among older adults? How do they work?”\nLoneliness is a major public health issue that has become critical now due to increased social isolation and lockdowns in the current COVID-19 pandemic.\nTherefore, this manuscript reporting a rapid review of reviews on remote interventions and their effectiveness in tackling loneliness in older people is timely.\nI have the following comments on the manuscript.\nAbstract:\n\nIn systematic reviews, reporting of the sources of literature, selection criteria and publication period are important but these are missing in the abstract. The authors need to add this information in the methods section of the abstract. If however there were no strict criteria such as restrictions on publication dates, then these need to be mentioned. The population of interest was ‘older adults’ and their age is reported as 50 years and above. This means no upper age limit therefore some participants might be very old such as above 70 years of age and they could be called as ‘elderly’. The two terms i.e. older and elderly, are interpreted differently; therefore, the authors might like to change the term ‘older adults’ to ‘adults aged 50 years and above’ to avoid the confusion. The Results section should also provide a summary of data extracted about characteristics of studies, population and interventions. Determination of the effectiveness of the interventions was one of the objectives and authors have reported a mixed quantitative evidence of effectiveness in the results section but nothing about the effectiveness is reported in the conclusion. The authors should include concluding remarks on the effectiveness in the conclusion in the abstract.\nIntroduction: In the introduction section, the authors mention older adults over 70 years while in the abstract they report 50 years plus. The authors need to be consistent in reporting the term older people and years of age covered. The authors might like to describe the term ‘older adults’ including age in years included in the review in the methods section.\nThe authors have mentioned that ‘During the height of the COVID-19 pandemic these interventions were of limited…’. This suggests that the peak intensity of the COVID-19 pandemic is over, which it is not because there are second and third waves of COVID in different countries. For example, the UK is currently going through a very serious second wave of COVID-19 and strict lockdown is in place across the country. Therefore, the authors need to change the above statement such as ‘during the height of the first wave of COVID-19 in 2020...’.\nMethods: Different researchers were involved in the screening and shortlisting of articles but no information on the interrater reliability/agreement is reported. Reporting of a relevant statistic such as the Kappa static will be helpful.\nResults: The results section includes some paragraphs such as the ‘Theory selection and setting up the QCA’, ‘Developing a data table’ and ‘Truth table’. These paragraphs describe the theory and methodological procedures; hence, these should be reported in the methods section.\nDiscussion: The authors might like to refer to some recent meta-analyses on the effectiveness of interventions to tackle loneliness, which show that digital interventions are not effective in adults aged 70 years and above (Shah et al., 20201) and especially video calls (Noone et al., 20202).\nConclusion: The conclusion section is more about the inclusion of the processes and principles suggested in the QCA. The authors need to include their overall inference about the effectiveness of different types interventions reviewed in the population of interest.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Partly\n\nIs the statistical analysis and its interpretation appropriate? Partly\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [ { "c_id": "6486", "date": "28 Apr 2021", "name": "Dylan Kneale", "role": "Author Response", "response": "Response to Reviewers. We thank the Reviewer for his constructive comments, which has given us the opportunity to improve the paper. We have addressed the comments and have revised the manuscript accordingly. Our point-by-point response is included below and we hope that we have satisfied the concerns of the reviewer. Response to Dr Shah: Abstract: In systematic reviews, reporting of the sources of literature, selection criteria and publication period are important but these are missing in the abstract. The authors need to add this information in the methods section of the abstract. If however there were no strict criteria such as restrictions on publication dates, then these need to be mentioned. The population of interest was ‘older adults’ and their age is reported as 50 years and above. This means no upper age limit therefore some participants might be very old such as above 70 years of age and they could be called as ‘elderly’. The two terms i.e. older and elderly, are interpreted differently; therefore, the authors might like to change the term ‘older adults’ to ‘adults aged 50 years and above’ to avoid the confusion. The Results section should also provide a summary of data extracted about characteristics of studies, population and interventions. Determination of the effectiveness of the interventions was one of the objectives and authors have reported a mixed quantitative evidence of effectiveness in the results section but nothing about the effectiveness is reported in the conclusion. The authors should include concluding remarks on the effectiveness in the conclusion in the abstract. Introduction: In the introduction section, the authors mention older adults over 70 years while in the abstract they report 50 years plus. The authors need to be consistent in reporting the term older people and years of age covered. The authors might like to describe the term ‘older adults’ including age in years included in the review in the methods section. The authors have mentioned that ‘During the height of the COVID-19 pandemic these interventions were of limited…’. This suggests that the peak intensity of the COVID-19 pandemic is over, which it is not because there are second and third waves of COVID in different countries. For example, the UK is currently going through a very serious second wave of COVID-19 and strict lockdown is in place across the country. Therefore, the authors need to change the above statement such as ‘during the height of the first wave of COVID-19 in 2020...’. Thank you for your comments and helpful suggested revisions to the abstract. We have included some further detail about the methods employed, within the confines of the word limit. We have added the fields which the 11 databases covered (‘from the fields of health, social care, psychology and social science). We have also added the selection was guided by our PICOS criteria. Thank you for pointing out the inconsistencies with regard to the age groups covered in the manuscript. The review included research studies with populations aged 50 years and above, whereas the UK Government guidance aimed at protecting older adults during the first wave of the pandemic was targeted at those aged 70 years and above. Thank you for helping us to clarify the time period with your wording about the first wave. We have changed the wording of the introduction to: ‘During the height of the first wave of the COVID-19 pandemic in 2020, millions of people aged 70 years and over were advised to avoid social contact with those outside their household. with older age identified as a risk factor for poorer COVID-19 prognosis (Mueller, McNamara et al. 2020).’ We have also changed the wording in the abstract, although the word limit has restricted what we could add here: ‘During the COVID-19 pandemic ‘social distancing’ has highlighted the need to minimise loneliness and isolation among older adults (aged 50+).’ With regard to the use of the term ‘older adults’, we feel that this term does encompass the full range of ages from 50 years and upwards. The term is commonplace in our research groups and we would prefer to keep it as it is, as many people in their 50s and 60s experience poor health and life transitions at an earlier age, meaning that they live with age-related conditions from 50+. As recommended, we have ensured that the term ‘older adults’ is defined in the introduction and at the beginning of our methods section, so that the reader is clear about the population in our paper, and is used consistently throughout. As such, we have changed all instances of ‘older people’ to older adults’. Introduction: ‘Here we use a broad definition of ‘older’ adult, defined as those aged 50+, which captures those in middle age who may be nearing or experiencing age-related transitions, such as retirement or unpaid caring, or living with age-related long term conditions.’ Methods: ‘For the purposes of this review, we define ‘older adults’ as those aged 50 years and above.’ Methods: Different researchers were involved in the screening and shortlisting of articles but no information on the interrater reliability/agreement is reported. Reporting of a relevant statistic such as the Kappa static will be helpful. We have now reported the level of agreement (93%) in the manuscript. Results: The results section includes some paragraphs such as the ‘Theory selection and setting up the QCA’, ‘Developing a data table’ and ‘Truth table’. These paragraphs describe the theory and methodological procedures; hence, these should be reported in the methods section. The theory has now been moved to the methods section. However, as the data table and truth table sections also include results, and provide a description of how to interpret the data presented, we think that these sections should remain in the results. As QCA is a relatively new method applied to systematic reviews, we believe that having this explanation alongside the results will provide clarity for the reader. Discussion: The authors might like to refer to some recent meta-analyses on the effectiveness of interventions to tackle loneliness, which show that digital interventions are not effective in adults aged 70 years and above (Shah et al., 20201) and especially video calls (Noone et al., 20202). Thank you for this suggestion, which has enabled us to improve the Discussion section. We have added the following paragraph at the end of the ‘Gaps in the evidence’ section: ‘Since this overview was published, two further systematic reviews have been published in the area examining the role of digital technologies (Noone, McSharry et al. 2020, Shah, Nogueras et al. 2020); both have suggested that the evidence on the effectiveness of certain forms of remotely delivered interventions is inconclusive. These inconclusive findings may reflect issues with the intervention approach and its implementation, but may also reflect the reality that evidence in this area is challenging to synthesise and characterised by heterogeneity in study design (with a preponderance of weak or flawed designs) and heterogeneity in outcomes that makes drawing conclusions challenging. For example, some of the interventions included in recent meta-analyses and in our own review were characterised as exhibiting negligible intervention impacts when considering post-test outcome scores alone, although significant changes between post-test and baseline scores suggest that a meta-analysis of change scores should also be considered in future. Furthermore, given that the majority of studies in this area do not implement robust RCT designs, reviews that place stricter inclusion criteria on the study design may only capture a narrow slice of the evidence base. As reflected in the protocol, our own review set out to examine effectiveness in the anticipation of extant meta-analyses in the field, although at the time of searching (April 2020), no eligible review had undertaken meta-analyses.’ Conclusion: The conclusion section is more about the inclusion of the processes and principles suggested in the QCA. The authors need to include their overall inference about the effectiveness of different types interventions reviewed in the population of interest. Thank you for highlighting this. We have added the following text to the end of the first paragraph of the ‘Further research and conclusions’ section: ‘Our original intention had been to examine the effectiveness of these approaches, although due to the heterogeneity in study design and the absence of existing meta-analyses in the literature at the time of searching, we were unable to do this directly. Instead in our QCA analyses, we identified studies and qualitatively allocated these into successful and unsuccessful groups based on their effect size and explored common characteristics of successful studies; we consider this to a be prudent way of mediating a need for a rapid evidence to inform policy with the need to implement robust and transparent methods to synthesise this evidence. As discussed, this literature has been developed further since the present review was completed (Gorenko, Moran et al. 2020, Noone, McSharry et al. 2020, Shah, Nogueras et al. 2020, Dubé, Paquet et al. 2021), and further progression in this area is being tracked through living maps of synthesised evidence (IPPO 2021).’" } ] }, { "id": "75713", "date": "25 Jan 2021", "name": "Jennifer Anne Chipps", "expertise": [ "Reviewer Expertise Mental Health and systematic reviews" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors did use the PRIMSA reporting guidelines. I do have a few concerns:\nThere are already a few umbrella reviews - these are not commented on and as to what his paper adds.\n\nWhich methodology for umbrella reviews were used?\n\nMore detail needs to be provided on the criteria of the primary papers extracted from the systematic reviews. E.g. What was the inclusion and exclusion criteria, and how was the quality of the primary papers assessed?\n\nIn methodology the ‘rapid’ process needs to be explained – e.g. what time period.\n\nDid the study include all residents in community and residential homes?\n\nTo address the question of effectiveness - only RCTS or QE studies should be included – there are qualitative papers in the synthesis which makes this confusing.\n\nSocial isolation and loneliness are separate concepts – need to be defined – this also related to the broad heterogenous measures included.\n\nWhat are diverse population of older persons?\n\nThe ICA and QCA is interesting and a new addition to these umbrella reviews\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Partly\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [ { "c_id": "6487", "date": "28 Apr 2021", "name": "Dylan Kneale", "role": "Author Response", "response": "Response to Reviewers: We thank the Reviewer for her constructive comments, which has given us the opportunity to improve the paper. We have addressed the comments and have revised the manuscript accordingly. Our point-by-point response is included below and we hope that we have satisfied the concerns of the reviewer. Response to Professor Jennifer Chipps: 1. There are already a few umbrella reviews - these are not commented on and as to what this paper adds. Thank you for this comment, enabling us to highlight what our paper adds. We have stated in the Introduction that a ‘number of evidence reviews have highlighted the diverse range of interventions to alleviate loneliness amongst older adults in a variety of settings’. We included references for Cattan et al., 2005 and Victor et al., 2018 here. We have now added Jarvis et al., 2020, which had not been identified in our searches in April 2020, and Veronese et al., 2020, which had not been published until after this research was conducted. In addition, we noticed an error in the referencing of one umbrella review (Chipps et al) which was introduced at the wrong place in the document. At the end of the introduction, we have made it clearer that our paper adds the explicit consideration of particular interventions (befriending, social support and low intensity psychosocial interventions) to address loneliness and social isolation, together with the innovative use of Intervention Component Analysis and Qualitative Comparative Analysis. ‘This rapid review examines evidence specifically on whether befriending, social support, and low intensity psychosocial interventions delivered remotely can reduce social isolation or loneliness among older adults.’ ‘(iii) Generate new understandings on how interventions work and which core components and processes are associated with successful interventions, using the innovative methods of Intervention Component Analysis and Qualitative Comparative Analysis.’ 2. Which methodology for umbrella reviews was used? We have added the following text to the first paragraph of the Methods section: Although broad frameworks for conducting overviews exist (Smith, Devane et al. 2011), specific guidance that can be used in a directive way to carry out overviews is lacking. Nevertheless, we followed elements of practice recommended by Caird and colleagues (2015) in balancing some of the challenges of conducting reviews of reviews with the need to produce policy-relevant evidence at speed in the context of the COVID-19 pandemic; the present review was developed over a four week period in April-May 2020. We also examined how existing reviews of reviews in the area, and particularly a review conducted by Chipps and colleagues (2017), navigated the challenge around differences between the scope of a systematic review and the scope of the review of the reviews. 3. More detail needs to be provided on the criteria of the primary papers extracted from the systematic reviews. E.g. What was the inclusion and exclusion criteria, and how was the quality of the primary papers assessed? Thank you for pointing out that this should be included. We have added the following text to the ‘Study selection and data extraction’ and ‘Critical appraisal’ sections: ‘Once eligible reviews had been identified, primary papers were extracted if they met the criteria in line with the review inclusion and exclusion criteria stated above. That is: Population: older adults, located in the community or residential care, socially isolated or lonely, or at risk of social isolation or loneliness. Intervention: interventions that sought to reduce levels of social isolation or loneliness, through strengthening individuals’ social contacts and social relationships. Interventions were delivered on a one-to-one basis (e.g. befriending), or as remote group-based interventions (e.g. discussion groups). Comparator/control: studies with most forms of control group (randomised and non-randomised) and those without a control group (pre-post designs). Outcomes: measures of social isolation or loneliness, including bespoke and proxy measures, such as social connectedness.’ ‘The quality of the primary studies was reported where it had been assessed by the review authors. Not all reviews included a quality assessment of their included studies.’ 4. In methodology the ‘rapid’ process needs to be explained – e.g. what time period. We have included the time period in the first paragraph of the Methods section: ‘the present review was developed over a four week period in April-May 2020.’ 5. Did the study include all residents in community and residential homes? We have added the following text into the Search strategy section: ‘1) Population: older and middle-aged populations aged 50+ years in the community and in residential settings.’ 6. To address the question of effectiveness - only RCTS or QE studies should be included – there are qualitative papers in the synthesis which makes this confusing. Thank you for highlighting this issue, which we have addressed through: editing the title of the manuscript to better reflect our research; The original title of the manuscript reflected the Protocol for the review which was to look at if, and how, interventions worked. However, at the time of searching (April 2020), no eligible review had undertaken meta-analyses, and we have clarified this and modified the title to reflect the content of the manuscript. adding a note on the study design in the Methods section: ‘As we were expecting some heterogeneity in the question being addressed by reviews, and expected this to be reflected in the design of primary studies included within reviews, we did not specify that source systematic reviews had to be confined to a particular study design. In line with previous reviews in the field, we expected studies measuring quantitative outcomes to be composed of single-group pre-post studies, non-randomised comparison studies, and randomised comparison group studies.’ 7. Social isolation and loneliness are separate concepts – need to be defined – this also related to the broad heterogenous measures included. We have defined social isolation and loneliness, as separate concepts, in the introduction and have added text to highlight this in the outcomes section of the ‘Inclusion and exclusion criteria’ section: We conceptualise loneliness as an emotional response by individuals when there is a ‘deficit between their desired and actual quality and quantity of social engagement and relationships 5 , p64’. Social isolation reflects the number of social contacts that people have (UK 2018), and people who are socially isolated tend to have social networks of low density that are not maintained through frequent engagement 6. Both loneliness and social isolation are conceptually distinct from living alone, the latter having limited utility as a proxy for either social isolation or loneliness 7 . However, we recognise that defining social isolation and loneliness is challenging, particular as researchers have used terms involving social relationships, including social isolation, loosely (Valtorta, Kanaan et al. 2016). Furthermore, while we recognise social isolation and loneliness as distinct concepts, here we explore both simultaneously as the COVID-19 pandemic and measures adopted to mitigate its spread have exacerbated both isolation and loneliness. 8. What are diverse population of older persons? Thank you for highlighting this lack of clarity. We have removed the word ‘diverse’ from the ‘Inclusion and exclusion criteria’ section, as it was unnecessary." } ] } ]	1	https://f1000research.com/articles/9-1368
https://f1000research.com/articles/10-325/v1	27 Apr 21	{ "type": "Research Article", "title": "Laughter down-regulates negative emotional arousal amongst friends", "authors": [ "Mimi Borrelli", "vikram sinha", "Sophie Scott", "vikram sinha", "Sophie Scott" ], "abstract": "Laughter is contagious, sensitive to social context, and can be used to mitigate negative emotional states. This experiment tested whether moments of transition from negative to positive affect were associated with more laughter if in familiar compared to unfamiliar company. 90 participants (47 females, mean age 20.61 years), either familiar (N=42) or unfamiliar (N=48) to the principle researcher, were randomly assigned to listen to 44 seconds of music which induced a fearful affect, positive affect or a neutral mood, followed by 30 seconds of infectious laughter (N=30/group). Filmed facial expressions were coded for four dependent variables (duration: half smile; full smile; laugh, and extent: peak mirth) of amusement in response to the laughter. Familiar participants fully smiled for longer than unfamiliar participants (F(1,84)=4.15, p=.045). There was an affect-familiarity interaction for peak mirth (F(2,84)=4.68, p=.01), time spent half smiling F(2,84)=5.00, p=.009), and fully smiling (F(2,84)=3.48, p=.035). Post hoc analyses revealed familiar participants exhibited greater peak mirth and smiled (half and full) for longer than unfamiliar participants in the fearful affect condition. Laughter and positive emotions may be used to moderate negative arousal more amongst people known to each other than amongst strangers.", "keywords": [ "Laughter", "Mirth", "emotional arousal", "social behavior" ], "content": "Introduction\n\nLaughter is an inherently social behaviour which is prevalent in interactions as wide ranging as tickling and conversations (Gervais and Wilson 2005; Provine 2001). Laughter is not automatic, rather highly modulated by social factors. Humorous material, for example, is rated more amusing when participants believe others have also found it amusing (Wimer & Beins, 2008).\n\nWithin interactions, laughter regulates mood and coordinates the behaviour of laughter through a group (Spoor and Kelly 2004; Deacon 1997; Provine 1992). Affect and laughter are induced in a listener both through the directly arousing acoustic properties of laughter, and indirectly as a conditioned positive affective social experiences (Owren and Bachorowski 2003). Laughter shows an in-group function: the act of laughing increases positive affect which further enhances the perceived pleasure of social interactions, promoting affiliative and cooperative behaviour (Owren and Bachorowski 2003; Martin 2007; Smoski and Bachorowski 2003). Participants laughed and smiled more, laughed longer, and rated humorous material more favourably when they heard in-group laughter as opposed to out-group laughter or no laughter at all (Platow et al. 2005).\n\nThe “undo” hypothesis theorises that positive emotions, including laughter and smiling, function to mitigate negative arousal that arises from stressful events (Levenson 1988). Cardiovascular arousal, including increased heart rate and blood pressure associated with a fearful film, dissipated more quickly when followed by happy films as compared to sad or neutral films (Fredrickson and Levenson 1998). Music-induced positive emotions preceded by the viewing of unpleasant pictures restored baseline cardiovascular and respiratory levels faster than did white noise (Sokhadze 2007).\n\nThe aim of the present study was to combine these two separate explorations of laughter and address the use of laughter to regulate negative emotional affective states amongst people known to each other. The study investigated whether more laughter and positive emotional expressions were produced by participants in familiar than unfamiliar company, in the context of transition from negative arousal to a more positive affect. Music was used to induce an affective state (positive or negative) or a non-affective state using non-arousing music as a control group. The music presentations were followed by presentation of infectious laughter. The hypothesis was that laughter produced in response to infectious laughter would be greatest following fearful affect, and would show sensitivity to social context, being greatest in familiar compared to unfamiliar company.\n\n\nMethods\n\n90 University students participated in the experiment (47 females, mean age 20.61 years (SD = 3.41, range 18-27). They provided written informed consent. Participants were recruited through personal contacts (N = 42 familiar), and through advertising (N = 48 unfamiliar).\n\nAmusement in response to the infectious laughter was the dependent variable. Manipulated mood (positive affect, negative affect, control) was the independent variable in a between-subjects design. Participants were randomly assigned, using a standardized table of random numbers on a 1:1:1 ratio, to one of the three experimental groups: 1) fearful affect; 2) positive affect; 3) neutral, ensuring 30 participants in each group. 17 participants were known to the experimenter in the fearful affect group, 11 in the positive affect group and 14 in the neutral group (Table 1). The assigned condition was written in pencil on the back of the printed consent forms.\n\nMusic induction excerpt\n\nA single suitable musical excerpt for each target mood appropriate for the target population was chosen from a large pool of potential stimuli using a pilot study (N = 6). The final fearful and positive affect excerpts were rated as those capable with inducing the strongest fearful and positive affect, with equal arousal ratings on an 11-point arousal scale (0–not arousing 10–highly arousing). The chosen stimuli were: Half Remembered Dream, by Hans Zimmer (Fearful affect), Monsier Verdoux Paris Boulevard, by Charlie Chaplin (positive affect). The control song Sleepy Noise, by Canton (neutral), was chosen based on its rating as non-arousing in the pilot study. Track tempo has been linked to arousal (Etzel et al. 2006) therefore the two emotionally arousing tracks were equated in tempi using Logic Studio’s Time Machine and AUpitch algorithms which leave the pitch intact. The fearful track reached a natural crescendo in 44 sec and pilot testing confirmed this was adequate to elicit the target emotional response, therefore equivalent sections in positive affect and control track were located and cut using Logic studio. The laughter tracks were included unexpectedly at the end of the audio tracks.\n\nLaughter tracks\n\nA pilot study was used to select the most contagious laughter sequences from a selection collaborated from the internet (www.freesound.org) and from live recordings from a social gathering recorded using a Dictaphone. Laughs from people of different sex and age were used to ensure ecological validity. The final selection contained 19 laughs which had been successful in eliciting laughter in all pilot participants.\n\nThis laughter track was edited onto the end of each musical excerpt, after a one second gap, using Logic Studio, to create three separate audio tracks for each experimental condition, each lasting 1 minute 15 seconds. All audio experimental stimuli were presented binaurally through headphones, and played off an iPod classic at a volume of 3.4 decibels.\n\nProcedure\n\nParticipants were tested individually. The testing location varied, but consistency was maintained through ensuring participants were always seated at a desk, in a quiet room with only the principle experimenter present. Participants were informed the experiment was an ‘Investigation into effects of acoustic stimuli on facial musculature’ and instructions, presented in written form, asked participants to listen to a short acoustic extract whilst their facial reactions were recorded on camera. The facial muscle movements of interest, they were told, were out of conscious and therefore they should not suppress or force any movement, just concentrate on the sounds they would hear. Participants consented to being filmed and blindfolded. Music induction procedures commonly ask participants to close their eyes to aid acoustical concentration (Etzel et al. 2006). Participants were instead blindfolded to alleviate participants of this responsibility. Once participants were blindfolded and wearing headphones, positioned ½ m from the Camera Recorder, the experimenter opened a numbered word document which revealed the assigned experimental condition and played the audio track appropriate to this condition. Facial expressions were continuously recorded. At the end of the audio, recording was suspended, headphones and blindfold were removed, and participants completed a questionnaire which asked them to “select one word that best represents the emotion elicited in you by the musical part of the acoustical excerpt” and whether they recognised the musical excerpts. Participants were then debriefed, and fully informed consent obtained.\n\nCoding\n\nThe videotaped recordings were digitalised and the 30 sec of behavioural responses from the onset of the laughter track were isolated for analysis. Playback was performed on iTunes Movies. Following previous research, amusement was measured using coding sheets along two dimensions: intensity and duration, which have shown maximum sensitivity to subtle differences in mirthful expressions (Platow et al. 2005). All facial image analysis coding was performed by independent observers (six for intensity of mirth, two for humour durations), who were blind to the experimental condition of participants and to the experimental hypotheses. Each observer was familiarised with two practice trials before analysis commenced.\n\nPeak Mirth\n\nA Mirth scale was created based on scales used in previous research. The Mirth Index created by Zigler and colleagues (Zigler, Levine, and Gould 1966) included five categories: negative response; no response; inhibited to a half or slight smile; full smile; laugh. The Mirth scale in this experiment adapted the Mirth Index (Zigler, Levine, and Gould 1966) by dividing the category ‘laugh’ into two; any indication of a laugh; and open mouth laugh. The categories and corresponding description, in brackets, were as follows:\n\n0. A negative response (a grimace)\n\n1. No response (blank expression, no indication of amusement)\n\n2. Any indication of a smile/amusement (open mouth, flared nostrils, trembling mouth\n\n3. A half smile (upturned corners of the mouth (symmetric or bilateral)\n\n4. A full smile (big and symmetrical lip curling, likely with teeth showing)\n\n5. Any indication of a laugh (audible sound, body movements (head or shoulders), distinctive breathing)\n\n6. Open mouth laugh (uncontrollable, full laughter response, clearly audible, big body movements)\n\nAll six categories were found easily discernible in a pilot study. Six observers were asked to mark for each participant the highest intensity level of mirth observed within the 30 seconds of participant recordings.\n\nMirthful durations\n\nThe total time each participant spent in: 1) half smiles; 2) full smiles; and 3) laughter, was used as measures of mirthful duration. Two coders identified the expressions according to a coding sheet of three categories adapted from previous research (Bachorowski, Smoski, and Owren 2001; Olson 1992; Vidulich and Bayley 1966; Deckers, Jenkins, and Gladfelter 1977). The description for category 1 Clear Smile is ‘clear upturned corners of the mouth’. Category 2 Big Grin is ‘clearly of higher intensity than clear smiles, clear bilateral movement and visible dimples’. Category 3 Laughter is ‘any audible obvious sound accompanying either a smile or grin’. The ‘laughter’ category was coded as ‘vocalised’ laughter. Eye movements were not included in coding facial expressions as the participants were blindfolded. Mirthful expressions were hand-timed using a stopwatch and measured in seconds (and not ‘events’).\n\nInter-observer agreement for all duration measures was high (rs = .9, p < .01), and for peak mirth it was reasonable (Kappa 55%). Average scores were calculated where observers disagreed. There were four dependent variable scores for each participant: 1) highest mirth achieved over the 30 seconds; and average time spent in 2) a half smile; 3) a full smile; and 4) laughing. A series of two-way independent-subjects analysis of variance (ANOVA) were run on each of the four variables, with induced-mood (fear, happiness, neutral) and familiarity (known, unknown) as the two between-subjects factors. Post-hoc tests were performed using the Bonferonni method. The effect sizes were calculated by hand using the mean difference equation (ES = Xi-Xj).\n\n\nResults\n\nParticipant demographic details and group compositions are above. Mean age, gender, and familiarity of participants, did not significantly differ between the three experimental groups. No participants recognised the musical part of the excerpts presented. Self-reported emotions were consistent with intended mood induced in each of the three conditions; negative and arousing emotions in the fearful aroused condition, positive emotions in the happy condition, mostly relaxing, though also some reports of positive and negative emotions in the control condition. Some participants reported that the laughter made them feel uncomfortable. Facial expressions of participants were generally reliably categorized on the mirth scale by observers. Observers expressed particular difficultly when trying to classify faces of participants who appeared to be stopping themselves laugh, where participants were seen to flare nostrils, bite lips, raise eyebrows and fidget or display other behaviours not enlisted in the mirth scale.\n\nThe peak mirth scores of familiar and unfamiliar participants did not significantly differ, F(1, 84) = 1.73, p < .05, and there was no significant effect of mood-induced, F(2, 84) = 1.59 p > .05. There was a significant interaction between the affect and familiarity, F(2, 84) = 4.68, p = .01 (Figure 1). Post-hoc comparisons revealed that familiar participants had significantly greater peak mirth scores (M = 3.27, SD = 1.34) than unfamiliar participants (M = 1.97, SD = 1.46) in the fearful affect condition (Figure 1). The size of this effect is 1.05.\n\nError bars represent the standard error of the mean. There is a significant interaction in all three dependent variables where familiar participants have higher peak mirth ratings, longer time spent smiling (full and half smiles) than unfamiliar participants in the fearful affect condition. Time spent laughing is not shown as results for this dependent variable were not significant.\n\nMood manipulations had no significant effect on subsequent time spent in half smiles, F(2, 84) = .576, p > .05. Duration of half smiles did not differ significantly between unfamiliar and familiar participants, F(1, 84) = 1.86, p > .05. There was a highly significant interaction between familiarity and affect, F(2, 84) = 5.00, p = .009 (Figure 1). Post-hoc analysis revealed that in the fearful affect condition familiar participants displayed half smiles for significantly longer (M = 24.36, SD = 9.81) than unfamiliar participants (M = 9.39, SD = 13.30), effect size 1.21 (Figure 1).\n\nFamiliar participants displayed full smiles for significantly longer (M = 11.56, SD = 11.13) than unfamiliar participants (M = 6.43, SD = 9.73), F(1, 84) = 4.15, p = .045, the size of this effect was small, .28. There was no significant effect of manipulated mood on subsequent time spent in full smiles, F(2, 84) = 2.33, p > .05. There was a significant interaction between familiarity and affect, F(2, 84) = 3.48, p = .035 (Figure 1). Post-hoc analysis revealed in the fearful affect condition familiar participants fully smiled for significantly longer (M = 17.07, SD = 11.44) than unfamiliar participants (M = 5.59, SD = 10.35). The effect size of this pairwise difference was 1.15 (Figure 1).\n\nFamiliar participants did not laugh significantly more than unfamiliar participants, F (1, 84) = .287, p > .05, and laughter durations did not significantly differ across condition, F (2, 84) = 3.01, p > .05. There was no significant familiarity vs affect interaction, F(2, 84) = 1.76, p > .05\n\n\nDiscussion\n\nThe present study asked whether laughter produced in the context of negative emotions was modulated by social context. The main finding was a significant affect-familiarity interaction in three of the four dependent variables. In the fearful affect condition participants familiar to the experimenter had significantly greater peak mirth scores, spent longer smiling (half and fully) in response to the infectious laughter than participants unfamiliar to the experimenter. Familiar participants also laughed for longer than unfamiliar participants in the fearful affect condition, but the familiarity-affect interaction was not significant. This is perhaps because fewer participants laughed overall in this condition, making the interaction underpowered.\n\nThis experiment was designed to combine previous work showing that laughter is both sensitive to social group and can regulate affective states within interactions. Laughter is greater amongst members of a group (Smoski and Bachorowski 2003) and is increased when participants hear in-group laughter (Platow et al. 2005) or when further affiliation with one’s experimental partner is desired (Grammer and Eibl-Eibesfeldt 1990). Additionally, positive affect, including smiles and laughter, down-regulate negative arousal arising from stressful events. This has been demonstrated in stressful events ranging from a forced speech task (Fredrickson et al. 2000), a fearful film (Fredrickson and Levenson 1998), viewing unpleasant pictures (Sokhadze 2007). The findings presented here uniquely demonstrate that mirthful expression following a transition from negative to positive arousal is greater when in company of in- compared to out-group, members.\n\nAlthough it was theorised that the significant interaction in the fearful affect condition was driven by more laughter amongst participants who knew the experimenter, it could be driven by less laughter in participants unknown to the experimenter. Graphical representation data of the dependent variables suggest known participants express more amusement in the fearful condition. However, some participants reported that the laughter made them uncomfortable. Laughter itself is a universal laughter-provoking stimulus (Provine 1992) reported by listeners to induce in them a positive affect (Szameitat et al., 2009). However, it can be heard in a negative way, especially in people who are “gelatophobic” and fear being laughed at (Ruch, Hofmann, and Platt 2015; Ruch et al. 2014). Perhaps the unfamiliar participants, who were blindfolded and in the company of a stranger, were more likely to perceive laughter negatively in the fearful affect condition.\n\nPrevious work suggests that laughter is greater amongst members of a group compared to strangers (Smoski and Bachorowski 2003; Platow et al. 2005; Grammer and Eibl-Eibesfeldt 1990). In this experiment, however, time spent fully smiling was the only dependent with a significant effect of familiarity; familiar participants fully smiled more than the unfamiliar participants. Familiar company only increased the duration of half smiles and extent of mirth in the context of negative emotions. Similarly, although laughter has been found to accompany the transition from negative-to-positive affect (Fredrickson et al. 2000; Fredrickson and Levenson 1998; Sokhadze 2007; Sroufe and Wunsch 1972), there was no significant effect of “affect” alone. These results suggest that the strongest stimulus to laugh and express is when participants are amongst familiar company and experience a transition from a fearful to positive affective state. This is a stronger stimulus to laugh that just being in familiar company or just experiencing transition from a negative-to-positive affect.\n\nHumour is involved in the formation, maintenance, and regulation of close interpersonal relationships (Lefcourt 2001; Shiota et al. 2004). Interactions associated with laughter increase feelings of closeness and attraction to ones’ confederate more than do equally enjoyable but non-humorous experiences (Fraley and Aron 2004). Positive emotions and laughter in humorous communications reinforce mutual feelings of affection, strengthen attachment, and contribute to greater relationship satisfaction (Smoski and Bachorowski 2003). Couples in highly satisfied marriages are better able to regulate negatively aroused states during conflictive interactions using positive affect, including laughter, affection and humour, than couples in less satisfied marriages (Gottman and Levenson 1992; Levenson and Gottman 1983; Carstensen, Gottman, and Levenson 1995) and have greater degrees of marital stability six years later (Gottman et al. 1998). Instructions to suppress negative emotions when discussing negative topics disrupted communication, inhibited relationship formation, and increased the blood pressure of the suppressor’s partner (Butler et al. 2003). It follows that laughter, a communicative group-sensitive behaviour, may have functional and evolutionary role in downregulating negative affective states in familiar company to facilitate successful group cohesion and group bonding. There is likewise less functional a role for laughter amongst strangers when faced with stressful situations.\n\nThe limitations of this experiment include the use of a blindfold. The involvement of eyes in smiles is important to distinguish between Duchenne (“emotional”) and non-Duchenne (“social”) smiles when coding smiles in social interaction (Ekman, Davidson, and Friesen 1990). It possible that the smiles recorded in this experiment were both “social” and “emotional” smiles. However, since participants were not directly interacting socially with a companion, “social” smiles were thought to be negligible. Participants were positioned in-front of a camera which could make participants feel self-conscious, and thereby inhibit emotional expressions.\n\nParticipants were blindfolded to both aid concentration on acoustic stimuli, and to block out the distraction of a camera. The benefits of using a blindfold for these purposes was thought to outweigh the benefit of including the eyes in rating of degrees of mirth. Another difficulty was found when coding faces for amusement in participants who appeared to be withholding laughter, and often used facial muscles other than the mouth. Future studies may benefit from including an additional “withholding laughter” category on mirth coding scales. “Familiarity” was coded categorically as unfamiliar or familiar, but it is recognized that this variable varies continuously. Future experiments could focus on whether the degree of familiarity influences the degree of mitigation from negatively arousing states. A strength of this experimental design was the use naturalistic methodology to evoke laughter, a sensitive emotion notoriously difficult to evoke in experimental situations. The design also included induction of a positive affective state which demonstrated the humorous response was specific to negative arousal as opposed to arousal in general as has been theorized (Berlyne 1972).\n\nOne interesting alternative explanation to the findings in this experiment relates to the ‘relief’ hypothesis of laughter (Ramachandran, Blakeslee, and Sacks 1998; Hayworth 1928; Keltner and Bonanno 1997). This hypothesis states that laughter accompanies the elicitation of positive affect, namely relief, following a negative state. The relief hypothesis can also provide unifying framework explaining why the focus of jokes worldwide tends to centre of taboo topics and slapstick (Freud 1989; Kotthoff 2006; Gervais and Wilson 2005). Future experiments will need to tease out these two alternative explanations of laughter.\n\n\nConclusion\n\nIn summary mirth intensity, smiles (half and full) were greater in known compared to unknown participants following induction of a fearful affect. This positive affect can be used to regulate negative affective states amongst people known to each other. The stimulus to laugh may be highest when in familiar companies and following a negatively arousal situation.", "appendix": "References\n\nBachorowski J-A, Smoski MJ, Owren JO: The acoustic features of human laughter. J. Acoust. Soc. Am. 2001; 110: 1581–97. PubMed Abstract \| Publisher Full Text\n\nBerlyne DE: Humor and its kin. The psychology of humor: Theoretical perspectives and empirical issues. 1972:43–60.\n\nButler EA, Egloff B, Wlhelm FH, et al.: The social consequences of expressive suppression. Emotion. 2003; 3: 48. PubMed Abstract \| Publisher Full Text\n\nCarstensen LL, Gottman JM, Levenson RW: Emotional behavior in long-term marriage. Psychol Aging. 1995; 10: 140. PubMed Abstract \| Publisher Full Text\n\nDeacon T: Evolution and intelligence: beyond the argument from design. The origin and evolution of intelligence. Boston: Jones and Bartlett; 1997; 103–36.\n\nDeckers L, Jenkins S, Gladfelter E: Incongruity versus tension relief. Motiv Emot. 1977; 1: 261–72. Publisher Full Text\n\nEkman P, Davidson RJ, Friesen WV: The Duchenne smile: Emotional expression and brain physiology: II. J Pers Soc Psychol. 1990; 58: 342. PubMed Abstract\n\nEtzel JA, Johnsen EL, Dickerson J, et al.: Cardiovascular and respiratory responses during musical mood induction. Int J Psychophysiol. 2006; 61: 57–69. PubMed Abstract \| Publisher Full Text\n\nFraley B, Aron A: The effect of a shared humorous experience on closeness in initial encounters. Pers Relatsh. 2004; 11: 61–78. Publisher Full Text\n\nFredrickson BL, Levenson RW: Positive emotions speed recovery from the cardiovascular sequelae of negative emotions. Cogn Emot. 1998; 12: 191–220. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFredrickson BL, Mancuso RA, Branigan C, et al.: The undoing effect of positive emotions. Motiv Emot. 2000; 24: 237–58. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFreud S: Jokes and their relation to the unconscious. WW Norton & Company; 1989.\n\nGervais M, Wilson DS: The evolution and functions of laughter and humor: A synthetic approach. Q Rev Biol. 2005; 80: 395–430. PubMed Abstract \| Publisher Full Text\n\nGottman JM, Coan J, Carrere S, et al.: Predicting marital happiness and stability from newlywed interactions. J Marriage Fam. 1998: 5–22. Publisher Full Text\n\nGottman JM, Levenson RW: Marital processes predictive of later dissolution: behavior, physiology, and health. J Pers Soc Psychol. 1992; 63: 221. PubMed Abstract \| Publisher Full Text\n\nGrammer K, Eibl-Eibesfeldt I: The ritualisation of laughter. Natürlichkeit der Sprache und der Kultur. 1990; 18: 192–214.\n\nHayworth D: The social origin and function of laughter. Psychol Rev. 1928; 35: 367. Publisher Full Text\n\nKeltner D, Bonanno GA: A study of laughter and dissociation: distinct correlates of laughter and smiling during bereavement. J Pers Soc Psychol. 1997; 73: 687. PubMed Abstract \| Publisher Full Text\n\nKotthoff H: Gender and humor: The state of the art. J Pragmat. 2006; 38: 4–25. Publisher Full Text\n\nLefcourt HM: Humor: The psychology of living buoyantly. Springer Science & Business Media; 2001.\n\nLevenson RW: Emotion and the autonomic nervous system: A prospectus for research on autonomic specificity.1988.\n\nLevenson RW, Gottman JM: Marital interaction: physiological linkage and affective exchange. J Pers Soc Psychol. 1983; 45: 587. PubMed Abstract \| Publisher Full Text\n\nMartin RA: The psychobiology of humour and laughter. In.: The psychology of humour: an integrative approach. Burlington: Elsevier Academic Press; 2007.\n\nBorrelli MR, Scott S: Laughter down-regulates negative emotional arousal amongst friends. Zenodo. 2021. Publisher Full Text\n\nOlson JM: Self-perception of humor: Evidence for discounting and augmentation effects. J Pers Soc Psychol. 1992; 62: 369. Publisher Full Text\n\nOwren MJ, Bachorowski J-A: Reconsidering the evolution of nonlinguistic communication: The case of laughter. J Nonverbal Behav. 2003; 27: 183–200. Publisher Full Text\n\nPlatow MJ, Alexander Haslam S, Both A, et al.: “It’s not funny if they’re laughing”: Self-categorization, social influence, and responses to canned laughter. J Exp Soc Psychol. 2005; 41: 542–50. Publisher Full Text\n\nProvine RR: Contagious laughter: Laughter is a sufficient stimulus for laughs and smiles. Bull Psychon Soc. 1992; 30: 1–4. Publisher Full Text\n\nProvine RR: Laughter: A scientific investigation. CITY, STATE: Penguin; 2001.\n\nRamachandran VS, Blakeslee S, Sacks OW: Phantoms in the brain: Probing the mysteries of the human mind. William Morrow New York. 1998.\n\nRuch W, Hofmann J, Platt T: Individual differences in gelotophobia and responses to laughter-eliciting emotions. Pers Individ Dif. 2015; 72: 117–21. Publisher Full Text\n\nRuch W, Hofmann J, Platt T, et al.: The state-of-the art in gelotophobia research: A review and some theoretical extensions. Humor. 2014; 27: 23–45. Publisher Full Text\n\nShiota MN, Campos B, Keltner D, et al.: Positive emotion and the regulation of interpersonal relationships. The regulation of emotion. 2004: 127–55.\n\nSmoski M, Bachorowski J-A: Antiphonal laughter between friends and strangers. Cogn Emot. 2003; 17: 327–40. PubMed Abstract \| Publisher Full Text\n\nSokhadze EM: Effects of music on the recovery of autonomic and electrocortical activity after stress induced by aversive visual stimuli. Appl Psychophysiol Biofeedback. 2007; 32: 31–50. PubMed Abstract \| Publisher Full Text\n\nSpoor JR, Kelly JR: The evolutionary significance of affect in groups: Communication and group bonding. Group Process Intergroup Relat. 2004; 7: 398–412. Publisher Full Text\n\nSroufe LA, Wunsch JP: The development of laughter in the first year of life. Child Dev. 1972: 1326–1344. PubMed Abstract\n\nVidulich RN, Bayley GA: A general field experimental technique for studying social influence. J Soc Psychol. 1966; 69: 253–63. PubMed Abstract \| Publisher Full Text\n\nZigler E, Levine J, Gould L: Cognitive processes in the development of children's appreciation of humor. Child Dev. 1966: 507–18. PubMed Abstract\n\n\nAppendices\n\nA mirth scale was used to measure the extent of amusement expressed by participants. Mirth scales are more objective than self-reports and thus not so susceptible to gender differences and demand characteristics. Observers assign a mirth score to the facial displays of participants. There is however no standardised mirth scale used by all observers, not even an agreed definition of Mirth (Martin, 2007), and typically new scales are generated to fit the data accordingly. A four-point scale is common (Pollio, Mers & Lucchesi, 1972) but higher gradations have been used (e.g. Darwin, 1872). Where these scales differ is mainly through the number of smiling categories (1 or 2), the number of laughing categories (1 or 2) and the inclusion of any negative categories. Video data was informally observed to get an understanding of the range and type of expressions shown by participants in this population. Since expressions appeared to be widespread, the full range of categories was thought best to achieve maximum sensitivity; two laughing intensities, two smiling intensities, one slight amusement, one neutral face and one negative face (see Table II). Category labels were defined based on observations and using definitions from the two scales shown in Table a. The same instructions were given to observers, to assign the highest category of mirth reached by each participant according to this scale, within the 30 sec playback period. Practice on two trials to familiarise coders with the technique was carried out beforehand, following previous work (Whor & Schwarting, 2007).\n\nTo be filled out by participant:\n\nTo be filled out by experimenter:" }	[ { "id": "140601", "date": "08 Jul 2022", "name": "Luísa Soares", "expertise": [ "Reviewer Expertise clinical psychology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper is interesting, and innovative. It also has scientific support to enrich the clinical psychologists in science and in practice. The procedures are very well presented and analyzed in the results and discussion.\nThe theoretical review is very updated and with good classical literature. The references are well presented. However, the English needs minor review.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "145984", "date": "16 Aug 2022", "name": "Adrienne Wood", "expertise": [ "Reviewer Expertise Nonverbal behavior", "social interaction", "laughter" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper presents an ambitious study that incorporates several hypotheses about laughter: 1) That people will laugh more with a friend vs a stranger, 2) That people will laugh more following a mild negative mood induction, as a sort of “undoing” of that negative mood, and 3) That the “undoing” effect will be moderated by being in the presence of a friend or stranger. These hypotheses are extremely interesting and merge ideas about laughter as social signals with ideas about laughter as emotion regulation.\nThe study design has several limitations that the authors need to address in the discussion more fully. The most important, in my mind, is that this turned out to not be a study about laughter production because the participants did not do much laughing. The key dependent variables are measures of smiling. And yet the introduction focuses on laughter and the discussion begins by saying “The present study asked whether laughter produced in the context of negative emotions was modulated by social context.” It is more accurate to say that the study is about whether hearing laughter after a negative mood induction has behavioral consequences that depend on social context. Similarly, the title might be more accurate as “Hearing laughter down-regulates...” rather than “Laughter…” because the latter implies the participants were laughing. There are other methodological limitations, described below, that are also worth mentioning in the discussion. For instance, it is unclear with the current design whether the participants’ mirth displays are responses to the laughter stimuli, to the mood manipulation, or both. Finally, my biggest concern about the analyses that isn’t an issue of interpretation is the use of ANOVA for a categorical outcome variable, Peak Mirth.\n\nIntroduction:\nThe introduction is quite short and all the references are at least 15 years old. I recommend incorporating more recent work.\n\nThe paragraph about the “undoing” hypothesis confuses laughter—which you established in the previous paragraph as being a highly social behavior—with positive emotions. For instance, the Fredrickson and Levenson (1998) paper is not about laughter undoing negative arousal; it’s about happiness undoing arousal. It’s not directly relevant here.\n\nMethod:\nThe familiar and unfamiliar participants were recruited through different channels, and may therefore differ systematically in any number of sociodemographic or personality dimensions. People tend to be friends with similar others, so is it possible that the “familiar” participants are all more similar in personality to the experimenter, and may share a tendency towards positive affect? This represents a confound in the study design.\n\nWhat did you do to reduce experimenter demand, given that the experimenter had to know which condition the participants were in (familiar vs unfamiliar)? The experimenter might have unintentionally acted differently with friends versus strangers.\n\nWhy did you only code the first 30 seconds of participants’ behavior when the laugh track played for 75 seconds?\n\nDid you use a correlation to quantify the inter-observer agreement? I don’t think this is the best approach because one person could be systematically biased to underestimate the duration, but still be highly correlated with the other rater.\n\nResults:\nPlease report the means and standard deviations for self-reported mood in the 3 emotion conditions (manipulation check).\n\nThe Peak Mirth dependent variable is categorical, so a regular ANOVA is not appropriate. For instance, see Jaeger (2008) for details.\n\nCould you more clearly explain the mean difference equation for calculating effect sizes? I assumed it was the difference between two groups’ means, but then I realized that isn’t right because the reported effect for Peak Mirth (1.05) is not the difference between means (3.27 and 1.97). it’s also not Cohen’s d, as far as I can tell.\n\nWe can’t conclude that the prolonged smiling in the familiar-fear condition (compared to unfamiliar-fear) condition is really about laughter down-regulating the fear. You would have needed a control condition in which people didn’t hear laughter after listening to fear-inducing music. Then you would know that the smiles are actually a response to the laughter-following-fear as opposed to just being a response to the fear (and not the laughter). In other words, the effect could equally be summarized as, “people smile more after hearing scary music if they are with a friend,” with no reference to the laugh track. Another solution would be to code the mirth displays of the participants while they listened to the music—then you can see whether the laughter indeed caused them to smile, or whether they were smiling more even while the fearful music was playing.\n\nDiscussion:\nAs I mentioned at the top, please modify the entire paper so it does not mislead the reader into thinking your results are about participant laughter production. For instance, examples from the discussion where you imply that the findings are all about participant’s laughter: “Although it was theorised that the significant interaction in the fearful affect condition was driven by more laughter amongst participants who knew the experimenter, it could be driven by less laughter in participants unknown to the experimenter.” And here: “These results suggest that the strongest stimulus to laugh and express is when participants are amongst familiar company and experience a transition from a fearful to positive affective state.”\n\nIn the discussion, if you’re going to state that (descriptively but not significantly) “familiar participants also laughed for longer than unfamiliar participants in the fearful affect condition”, then please report the supporting means and SDs in the results section.\n\nPlease clarify how your pre-recorded laughter induction is “naturalistic,” as you say in the discussion: “A strength of this experimental design was the use naturalistic methodology to evoke laughter, a sensitive emotion notoriously difficult to evoke in experimental situations.” Readers might object to this statement since it is a fairly unnatural situation the participants find themselves in, listening to decontextualized laughter while blindfolded.\n\nYou mention the “relief” hypothesis of laughter in the discussion. Could you say more about how this is different from the “undoing” hypothesis and where they make different predictions?\n\nMinor:\nThis line in the introduction is circular, with laughter coordinating the behavior of laughter: “Within interactions, laughter regulates mood and coordinates the behaviour of laughter through a group”.\n\nI think you need to change the “less than” sign to “greater than” in this sentence from the Peak Mirth results: “The peak mirth scores of familiar and unfamiliar participants did not significantly differ, F(1, 84) = 1.73, p < .05”.\n\nThe paragraph from the Discussion that begins “Humor is involved in the formation, maintenance…” feels more appropriate for the intro (plus the intro could use more meat anyway).\n\nI hope this feedback is useful. I look forward to seeing the next version of the paper and future work on this topic.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/10-325
https://f1000research.com/articles/10-321/v1	26 Apr 21	{ "type": "Research Article", "title": "The effect of morning versus evening administration of empagliflozin on its pharmacokinetics and pharmacodynamics characteristics in healthy adults: a two-way crossover, non-randomised trial", "authors": [ "Rana M. ElDash", "Mohamed A. Raslan", "Sara M. Shaheen", "Nagwa Ali Sabri", "Mohamed A. Raslan", "Sara M. Shaheen", "Nagwa Ali Sabri" ], "abstract": "Background: Empagliflozin is an SGLT2 inhibitor approved for use in patients with diabetes mellitus type 2 (DMT2) with or without other cardiovascular disease. Empagliflozin is taken once daily without rationale on the optimal timing for administration. This study aimed to determine the chronopharmacological effects of morning vs evening administration of empagliflozin (10 mg) in healthy Egyptian adults, by investigating the pharmacokinetics and pharmacodynamics parameters of empagliflozin depending on the intake time. Methods: An open label, sequential, two‐way crossover trial comprised of two periods with a washout period of 7 days. All participants received a single oral dose of empagliflozin (JARDIANCE ®; 10 mg film coated tablet) in the evening, and after a seven-day washout period, the morning. Pharmacokinetics parameters (primary endpoints: tmax (h), Cmax (ng/ml), AUC 0-t (ng.h/ml); secondary endpoints: AUC 0 to ∞(ng.h/ml)) were assessed. Method validation was done prior to injection in LC/MS/MS and samples were processed by Liquid-Liquid extraction. The pharmacodynamic profile (UGE 0-24) was determined after method validation (glucose hexokinase method). Results: Tmax increased by 35% in the evening phase compared to the morning phase, while Cmax decreased by -6.5% in the evening dose compared to the morning dose. Additionally, AUC0 to ∞ increased in the evening phase by 8.25% compared to the morning phase. The mean cumulative amount of glucose excreted (UGE (0-24)) increased by 43% in the evening dose compared to the morning dose Conclusion: Despite the difference in pharmacokinetics parameters between evening and morning doses, Cmax, AUC0-t, AUC 0-∞, didn’t differ on the bioequivalence level. In addition, as UGE (0-24) didn’t statistically differ, thus, we can conclude that there is no statistical significance between the morning and evening doses. Trial registration: Clinal Trials.gov, ID: NCT03895229 (registered on 29th March 2019).", "keywords": [ "Chronopharmacology", "Empagliflozin 10 mg", "Diabetes Mellitus", "Bioequivalence", "morning dose", "evening dose", "Pharmacokinetics", "Pharmacodynamic", "Circadian rhythm." ], "content": "Introduction\n\nDiabetes mellitus (DM) is one of the leading causes of death in the world. It has become one of the most critically medical and socially impacted diseases of the 21st century. Moreover, it is considered an epidemic disease with very high proportion of cases discovered every year. According to the World Health Organization (WHO), the prevalence of DM worldwide almost doubled from 4.7% to 8.5% between 1980 and 2014, reaching approximately 422,000,000 of diabetic adults in 2014, particularly in low-and middle-income countries like Egypt1. By 2040, around 642 million people will be diagnosed with DM2,3.\n\nChronopharmacology is the field of study concerned with the circadian rhythm of drugs regarding its pharmacokinetics/pharmacodynamics and pharmacological action; it also determines how the timing of the day may change the pharmacological action, pharmacokinetics and pharmacodynamics of the drug. Results of chronopharmacological studies are taken into practice under the umbrella of chronotherapy4. In addition, chronopathology proposes that any disease, including DM, can happen as a result of disruption of biorhythms5,6.\n\nThe pathogenesis of DM involves different types of models, which explain various mechanisms of its pathogenetic development. For many years, the glucocentric approach has shaped the main theory of DM development. The new multigene concept shifted the theory of glucocentricity, and as a result, it added a new pathway for treating DM; many theories are presented which may open up our understanding on how circadian rhythm affects DM5.\n\nCircadian rhythm influences the kidney; the glomerular filtration rate (GFR) and renal plasma flow, together with tubular secretion and absorption, work promptly in the active phase and decrease activity in the inactive phase. These mechanisms are controlled by a circadian clock, which dominates multiple cellular functions, such as the transcription and translation of proteins, the addition of phosphorous or acetyl group, and even the ubiquitylation of protein (post transitional). Moreover, kidney functions (electrolyte excretion, urine volume and regulation of blood pressure) abide by circadian variation7,8.\n\nThese various circadian etiologies affecting the development of DM necessitate the study of how chronopharmacological studies on antihyperglycemic drugs can affect the outcome of the treatment. This is the aim of personalized medicine. However, there are only a few available chronopharmacological studies to date available for antihyperglycemic drugs5.\n\nIn this study, we propose to investigate empagliflozin. Empagliflozin, which acts by inhibiting the sodium glucose co-transporter 2 (SGLT2) in the proximal tubules in the kidney, is indicated for two major isoforms (SGLT1 and SGLT2) in DM type 2, which are proposed for the sodium-glucose cotransporter (SGLT). SGLT2 is mainly expressed in the lumen of the small intestine and kidneys; SGLT2 takes part in the absorption/reabsorption of glucose induced by the sodium gradient across the cell membrane in the proximal tubules in the kidney9. Empagliflozin shows high improvement in glucose metabolism and, hence, its homeostasis. When added to standard care, it demonstrates, along with its anti-glycemic effect, a decrease in the patients’ mortality rate attributed to the cardiovascular disease(s), hospitalization for heart failure, all-cause hospitalization, and all-cause mortality10. As a result, in 2016, the FDA announced that empagliflozin is indicated for diabetic patients with heart failure11. In addition, in 2021, a new drug application is being investigated for empagliflozin in reducing risk of mortality or hospitalization and preserve kidney function in diabetic and non-diabetic with heart failure12,13. Moreover, it has a positive impact on glycemic control and reductions in weight and normalization of circadian blood pressure rhythm (non-dipping)9.\n\nEmpagliflozin (JARDIANCE ® 10 mg film coated tablet) is found in the market in two concentrations (10 or 25 mg), taken once daily with no rationale on the intake time between the morning versus the evening10,11.\n\nEmpagliflozin is swiftly absorbed after single escalating oral doses (0.5–800 mg) with a two phases decline. AUC0-∞ and Cmax are directly proportional with increasing doses. Moreover, in a few studies administration of empagliflozin with food resulted in halting the absorption, and in addition, the total quantity of glucose excreted increased by increasing the doses; it inhibited 40% of reabsorbed glucose at the single daily doses2.\n\nThe timing of drug intake can affect the circadian rhythm. For example, the blood pressure lowering drug Fimasartan shows pronounced blood pressure lowering (readjusting the dipping pattern) at night compared to morning dose. This is similar to our study drug, empagliflozin, which shows a provisional effect in restoring circadian blood pressure also at night in patients suffering from an imbalance in dipping blood pressure rhythm14,15.\n\nIn addition, since empagliflozin works on the kidneys’ proximal tubules, therefore, by examining the effect of inhibiting SGLT2 in the proximal tubules from a circadian rhythm point of view, it can add a new perspective in studying how the circadian rhythm can affect the timing of drug administration in the kidney.\n\nThe aim of this study is to examine the chronopharmacological characteristics of empagliflozin (10 mg film coated tablet) by comparing the antihyperglycemic effect (morning vs evening administration) on healthy adults, and to examine the influence of circadian rhythm on empagliflozin. Therefore, we examined for the first time the effect of the day and night dosing on the circadian rhythm for an antihyperglycemic drug, which may aid improving the proper use of this drug.\n\n\nMethods\n\nThis open label, sequential trial consisted of two periods with a seven-day washout period and took place between 2nd and 10th October 2018. Participants were selected 21–27 days before the trial between the 3rd of September to 26th of September 2018. Figure 1 includes the trial study design.\n\nThe trial was conducted at the Drug Research Center, Cairo, Egypt in compliance with Good Clinical Practice and in accordance with the International Conference of Harmonization (ICH) guidance on general considerations for clinical trials and the Declaration of Helsinki. The trial was approved by the Ethics Committee (IRB) of the Faculty of Pharmacy of Ain Shams University (no.203) and the Drug Research Center. The trial was registered in ClinicalTrials.gov (ID: NCT03895229) on 2nd April 2019. The trial was registered after the trial had been completed due to an administrative error. The authors confirm that all ongoing and related trials for this drug/intervention are registered.\n\nEach participant provided written informed ethic to participate in the trial prior to enrolment.\n\nParticipants were selected 21–27 days before the trial and were recruited according to eligibility criteria from the drug research center volunteers’ database and volunteer referrals. In addition, dietary regimens (meals) were supplied in accordance with the FDA and Food and Nutrition Board, and all participants received the standard total caloric intake/day of fats (20–35%), carbohydrates (45–65 %) and protein (10–35%)16.\n\nEligible participants were non-smokers with good age-appropriate health conditions, as established by medical history, physical examination, and results of biochemistry, hematology and urine analysis testing four weeks prior to the study (see section Laboratory tests). Eligible participants required normal blood pressure and pulse rate according to reference normal ranges.\n\nThe sample excluded any participants who had been subjected to known enzyme-inducers/inhibitors within 30 days prior to the start of the study, and participants who took any medication less than two weeks prior to the trial starting date. Participants susceptible to allergic reactions to empagliflozin, or any other condition that might interfere with drug absorption, including prior surgery of the gastrointestinal tract, gastrointestinal diseases were excluded. Those with renal diseases, cardiovascular diseases, hepatic diseases, hematological disease or pulmonary disease, having dehydration, hypotension, urinary tract infections and cases of abnormal laboratory values were rejected. Finally, participants who had donated blood or who had been in multiple dosing study requiring a large volume of blood (more than 500 ml) to be drawn within six weeks preceding the start of the study were also excluded.\n\nScreening assessments encompassed complete (past and present) medical history evaluation, physical examination and laboratory tests, namely: (1) biochemical tests of fasting blood sugar, serum urea, serum creatinine, serum glutamic-pyruvic transaminase, serum glutamic-oxaloacetic transaminase, cholesterol, triglycerides; high-density lipoprotein, low-density lipoprotein, hepatitis c virus antibody, human immunodeficiency virus, blood Group (A, B, AB, O) and RH-typing; (2) a complete blood count report; (3) a urine analysis report, which included physical examination, chemical examination and microscopical examination.\n\nThe participants were allowed to eat a standard meal containing carbohydrates, protein and fats according to the allowed ratios of standard calories per day (2000 kcal/day).\n\nThe participants entered the Drug Research Center one day before the treatment began, in which they received their morning or evening treatment doses by a resident physician and a clinical pharmacist.\n\nEach participant had to go to another room to receive the dose and then come back to the main room. Evening dose was given on the 2nd of October and morning dose was on the 10th of October 2018.\n\nIn order to increase patient compliance during the trial, incentives were offered, free meals (as above) and transportation after the end of the trial.\n\nAll participants received their evening does first, stayed for 24 hours to have their urine and blood tests; then came back 7 days later to receive their morning dose, and stayed for 24 hours for urine and bloods, then came back for a follow-up at 48 hours.\n\nEligible healthy participants received a single oral dose of empagliflozin (JARDIANCE ®; 10 mg film coated tablet; manufactured by Boehringer Ingelheim Pharma GmbH & Co. KG, Germany for Boehringer Ingelheim International GmbH, German) in the evening or morning17.\n\nIn the evening phase, at around 7 pm, the participants were administered the evening oral dose followed by dinner. After the washout period (7 days), at around 9 am, the participants then received the morning oral dose followed by breakfast.\n\nThe trial medication was administered with a full glass of water. The participants were asked to fast for at least 12 hours before the dose of empagliflozin (JARDIANCE ® 10 mg) and the dose was administered under close supervision of a medical investigator.\n\nPrimary pharmacokinetic endpoint was to determine the effect of morning versus evening doses on pharmacokinetics parameters: Tmax (h), Cmax (ng/ml), AUC 0-t (ng.h/ml), while secondary pharmacokinetics parameter was AUC (0 to ∞) (ng.h/ml)18.\n\nPharmacodynamic endpoint was to determine the cumulative UGE (cumulative amount of glucose excreted) over the 24 h in g/dl18.\n\nUrine samples were collected by urine collecting tube (1liter) for determination of UGE in g (urinary glucose excretion), which is the mean amount of glucose excreted in urine over the first 24 h after oral administration. Sampling intervals were 0 to 4, 4 to 8, 8 to 12 and 12 to 24 h after the administration of empagliflozin at the morning and evening doses.\n\nUrine samples were stored at -80 °C before being sent to the clinical laboratory for analysis. Analysis was performed by the glucose hexokinase enzymatic method for determination of glucose concentration19.\n\nCumulative UGE (mg) was calculated for each participant by multiplying the urine volume by the glucose concentration for each sampling interval in each period (morning or evening).\n\nSerial blood samples (0.5 ml) for determination of plasma empagliflozin concentrations were collected at 0 h pre-dose and then at 0.333, 0.667, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 4, 6, 8, 10, 12, 24, and 48 h for period one (evening dose) and period two (morning dose). Bioanalytical method validation was done by the authors; blood samples treated with EDTA were centrifuged at 3500rpm for 5 minutes to get a plasma supernatant.\n\nPlasma samples were analyzed by high performance liquid chromatography tandem mass spectrometry (HPLC- MS/MS) using an LC Agilent 1200 series and an Agilent 6410 Quadrupole mass spectrometer (Agilent Technologies, Inc., Santa Clara, CA) for determination of empagliflozin concentration in plasma. For preparation of plasma samples, 50ul of the internal standard dapagliflozin was added to an aliquot of 0.5ml plasma then vortex mixing was applied for 30 seconds. A liquid-liquid extraction by 2mls of diethyl ether-dichloromethane 60:40v/v was performed. After vortex mixing samples with the organic solvent for 2 minutes, samples were prone to phase separation at 3500rpm for 5 minutes in the centrifuge (Hermle Z 326K, Hermle Labortechnik GmbH, Wehingen, Germany). The clear supernatant was separated after centrifugation and evaporated under vacuum in the concentrator at 450C (Vacufuge® Plus, Eppendorf, Germany). Reconstitution of dry residue by the mobile phase (0.4% formic acid:Acetonitrile 17:83v/v) was performed prior to injection on LC/MS/MS. An aliquot of 5ul was injected on an isocratic system with a mobile phase composed of 0.4% formic acid: Acetonitrile 17:83% v/v and a C18 column as the stationary phase (Gemini C18 50 X 4.6mm, particle size 5um, Phenomenex Inc, Torrance, CA.) with a total run time of 3.1 minutes. Empagliflozin and dapagliflozin were determined by mass spectrometry in the multiple reaction monitoring mode (MRM). The mass to charge ratios (m/z) of precursor ions monitored for empagliflozin and dapagliflozin were 429.4 and 423.4 respectively with a common product ion m/z 207.1. The method of analysis showed a linear calibration range from 0.5 to 200 ng/ml with a correlation coefficient (r2=0.999) and average accuracy percent of 98.7%. Inter-day precision for quality control samples were below a percent relative standard deviation of 5.5% confirming a precision within the acceptable limits of validation.\n\nPharmacokinetics parameters of Empagliflozin were analyzed by Winnonlin™ software version 2.0 (Pharsight, California, Palo Alto, CA). Cmax, Tmax were calculated directly from the plasma concentration time curve (shown in Results section), while AUC 0-t and AUC0 to ∞. were computed using the linear trapezoidal rule.\n\nThe subjects were followed up during and after one day of the study. Results obtained from the laboratory tests (biochemical, complete blood picture report, and urine analysis report) were done prior to the study with only participants with results within the normal range allowed to participate in the study. In addition, medical history and physical examination were done and evaluated by a physician in the research center prior to the start of the study. Adverse effects were recorded by participants during the two periods (night and day).\n\nBased on a within subject variability of 18.7% for Cmax of empagliflozin20, a calculated sample size of 16 subjects was sufficient to acquire a study power of 80% at a level of significance of 5%21,22.\n\nAll subjects were tested for the pharmacokinetics and pharmacodynamics endpoints; analysis of the study was intention to treat. Diurnal and nocturnal variability from morning and evening doses were assessed independently for all the pharmacokinetics and pharmacodynamics parameters.\n\nPharmacokinetics primary endpoints (Cmax, AUC0-t) and secondary endpoint (AUC0-∞) were tabulated; one-way analysis of variance (ANOVA) was used, the data was assessed as mean difference between the two phases. Results were shown as P-value, geometric mean with 90% CI (confidence interval). Pairs of logarithmic transformed data of the primary and the secondary of the endpoints were analyzed using SAS® University edition statistics software (SAS®, USA) to determine a statistical difference between the morning and evening phases. Tmax was analyzed by Pairwise comparisons using the Wilcoxon signed-rank test. Median and range for both morning and night data and difference in median and P value were reported.\n\nPharmacodynamics parameter (cumulative amount of glucose excreted) was shown as P-value, geometric mean with 90% CI. Assessment was done by comparing mean difference of cumulative amounts of urine excreted over the 24 h for each phase (morning or evening) by one-way analysis of variance (ANOVA) for determining a statistical difference between the morning and evening phases using SAS® University edition statistics software. P-value of < 0.05 and 90% CI for a two-tailed test were used to assess the significant difference in the study hypothesis.\n\n\nResults\n\nIn total, 16 adult men participated in the study; median age was 26 years (range 18–55 years) with median body mass index of 24.285 kg/m2 (range 20–30 kg/m2). The baseline demographics of all study participants are shown in Table 1. All 16 participants completed the study (no drop out), and all results were included in the pharmacokinetics, pharmacodynamics.\n\nMean cumulative amount of glucose excreted from a single dose of empagliflozin (10 mg) over the 24 hours in the evening (Phase 1) and morning (Phase 2) doses are shown in Table 2 (Figure 2 and Figure 3). Mean UGE (0–24) for the evening dose (phase 2) was 69 (CV%=43.4), which was higher compared to the morning (phase 1), at 39 (CV%=41). Geometric mean results comparing the evening and morning doses for the log transformed UGE values was 116.7 (90% Cl 79.8-170.8; P=0.7317).\n\nEvening and morning doses of empagliflozin once daily were rapidly absorbed (in favor the morning dose) with peak median Tmax ranged between 2.5 (interquartile range (IQR) 1.25) to 1.625 (IQR 1.3) h post dose (P=0.063), reaching the maximum concentration between 125.60±30.7 (24.4%) to 117.9±32 (27.16%) g (P=0.324). The latter was followed by the slow elimination phase with elimination half-lives in the evening and morning doses of 7.2 (IQR 1.6) to 7 (IQR1.4) h, respectively (Table 3, Figure 4).\n\nIn addition, the total exposure of the drug in a form of the area under the curve (AUC0-t) between the morning and evening doses ranged from 888 (21.05%) to 960 (16.35%) (P=0.057). AUC0-∞ ranged between 899 (21.02%) to 980 (16.4%) (P=0.036) (Table 3, Figure 4). All the findings above suggests a linear pharmacokinetics relationship with time.\n\nIn addition, the geometric mean results of comparing log transformed Cmax, AUC0-T and AUC0-∞ for the evening and morning doses were 93.6 (90% Cl 83.707-104.82), 108.9 (90% Cl 101.274-117.297) and 109.823, (90% Cl 102.243-117.964), respectively.\n\nThe total exposure of the drug measured by AUC0-t relative to the extrapolated total AUC0-∞ of empagliflozin was slightly higher for evening than morning doses (98.776±0.541 vs 98.030±0.794).\n\nNo adverse events were detected from empagliflozin 10 mg in this study. The drug was well tolerated for all participants. Vital signs before, during and after the end of the study (blood pressure, pulse) were within normal ranges. Clinical and lab values were all evaluated to be within normal values.\n\n\nDiscussion\n\nChronopathology proposes that any disease, including DM, may occur as a result of disruption of biorhythms23. The effect of circadian clock on the kidney may affect the pharmacokinetics and/or pharmacodynamics of many drugs, especially those that exert their mechanism of action in the kidney7. Moreover, circadian clock proteins control most hormones, enzymes, and transport mechanisms related to glucose metabolism24. The following examples show different techniques on how circadian rhythm influence the kidney’s action: it has been described that renal function cycles over the 24 h and follows rhythmicity in its action, of note, GFR is influenced by many factors such as; systemic blood pressure, renal blood flow, afferent and efferent arteriolar resistance regulation, sympathetic system and hormones as renin and vasopressin25.\n\nFactors affecting GFR also affect other mechanisms within the kidney. Stimulation of the sympathetic nervous system influences renin secretion and renal sodium reabsorption (which can affect the action of our study drug)25. Previous studies showed that SGLT2 expression in the proximal tubules is increased in DMT2, which may suggest that further studies to be done on the expression of SGLT1 and SGLT2 at the proximal tubules in the kidney (in which empagliflozin inhibit) are required14.\n\nOur study examined the effect of the SGLT2i with empagliflozin, taking into account that the transport of solutes in and out of the kidney are controlled by the circadian clock. It has been shown that NH3 and SGLT1 are controlled by circadian oscillations proteins (Per 1 and BMA1), affecting the mRNA transcription26.\n\nOther studies determined the mechanism and the level of involvement of the circadian clock genes and circadian rhythms in the proximal tubule cellular Na+/H+ ex- changer 3 (NH3) transporter. In addition to other mutations in circadian clock proteins, rhythmic oscillations involved in NH3 activity are directly related to positive daily variations in sodium and water transport of the proximal cells27. NH3 expression is activated in the dark cycle and increases during a food intake26,28.\n\nAs sodium-hydrogen exchanger is affected by the circadian clock and, due to the link between empagliflozin (SGLT2i) and sodium-hydrogen exchanger, contributes to preventing heart failure. We recommend performing further studies on the effect of circadian rhythm on the action of SGLT2 inhibitors in specific and antihyperglycemic agents in general29.\n\nCircadian clock genes appear to be involved in every biological process in the human body. Currently, there is a lack of studies examining and determining the effect of chronopharmcology on antihyperglycemic drugs and drugs that exert their activity on the kidney. We recommend more chronopharmacological studies for antihyperglycemic drugs, either working on insulin secretion level or, as per our study, or drugs, which exert their mechanism of action on the kidney5,24.\n\nA few recent studies have started to address the relationship between chronopharmacology and antihyperglycemic drugs. One recent study examined the effect of administrating dapagliflozin on high-fat diet-induced obesity in mice; the results of the study were promising and suggest that dapagliflozin follows chronopharmacology as plasma glucose, insulin levels and adipose adipokines decreased in the light phase30.\n\nEmpagliflozin (JARDIANCE®) is available in two doses: 10 and 25 mg31. We examined the 10 mg dose. Empagliflozin can be taken once daily (without rationale on its timing) as oral bioavailability of empagliflozin is high (higher t½)2. In the current study, empagliflozin 10 mg once daily in the morning or evening was rapidly absorbed, reaching the peak tmax (Cmax) range between 2.5 and 1.625 h post dose, which similar to other previous studies on diabetic patients and healthy volunteers (1.5 to 2.1 h)2,32,33. Absorption followed by the slow elimination phase with approximately the same elimination half-life in the evening and morning doses (7.278–7.327 h), which appeared shorter compared to previous studies that reached up to 13 h2,18.\n\nThe results of the current study showed that the total exposure of the drug measured by AUC0-t relative to the extrapolated total AUC0-∞ of empagliflozin was slightly higher for evening than morning doses (98.776±0.541, 98.030±0.794). On the other hand, UGE (0–24) for the evening dose was 69 g, compared to the morning phase, which was quite low at 39 g. Comparing these results to previous studies examining once daily dose of empagliflozin 10 mg, UGE0–24 was 47.9 in healthy Caucasian and 50.6 g in healthy Japanese populations, respectively21,34. Moreover, in diabetic patients it ranged between 46.3 and 89.8 for the single dose to all doses of empagliflozin2.\n\nIn studies done on diabetic patients, UGE0–24 for the single dose of 10 mg ranged between 74.9 g and 77.9 g32,33. In this study, although the UGE0–24 for the evening dose was higher, it didn’t reach a statistically significant level35.\n\nMoreover, the study on healthy Japanese participants showed that as the exposure increased (Cmax and AUC0-∞), the UGE increased, which might correlate with our study findings (higher AUC0-∞, UGE0–24) favoring the night dose, except that Cmax didn’t differ between morning and evening doses21.\n\nThe reason behind the higher UGE (0–24) for the evening dose may be attributed to the short duration of the study or may result from the effect of food since the dinner meal is always higher in calories, compared to the morning meal. However, previous studies that examined the role of food on empagliflozin (50 mg) showed that UGE0–24 didn’t differ significantly between the fasted and fed state, with 71.7 g (13.6) and 75.9 g (17.9) mean (SD) respectively in healthy volunteers34. Moreover, another study, done on the 25 mg dose, showed a non-significant effect with food administration (geometric mean 84.04, 90% CI 80.86-87.34) for the AUC0-∞.36. As a result, due to the limited studies on the effect of food on the morning and evening doses from the pharmacokinetics and pharmacodynamics perspective, further studies may be required.\n\nDue to the above-mentioned functions of empagliflozin, we studied the effect of circadian rhythm and its time of administration regarding its pharmacokinetics and pharmacodynamics parameters, as we aim to achieve the best use of the drug, towards implementing personalized medicine.\n\nThis study was a pilot, done on healthy participants to limit confounders (other drugs or commodities) that can affect the study aim. Other studies performed on DM patients (their characteristics and physiological effects) are recommended to further explain if circadian rhythm can affect the pharmacokinetics and pharmacodynamics of the drug and thus efficacy of antihyperglycemic drugs. In addition, more studies can be performed addressing the role of pharmacogenetics changes and chronopharmacology.\n\n\nConclusion\n\nAlthough there was a difference in the overall exposure of the empagliflozin in the morning vs evening doses (P value was significant for AUC0-t and AUC0-∞), they all were within the bioequivalence range. The difference in the other empagliflozin pharmacokinetic parameters between the evening and morning doses were non-significant. In addition, (UGE0–24) was higher for the evening dose, but it didn’t reach a significant level. Taken together, the findings of the current study provide the first evidence that there is non-significant difference in the pharmacokinetics and pharmacodynamics effects between evening and morning dosing of empagliflozin 10 mf film-coated tablets.\n\n\nData availability\n\nDryad: Demographic data, https://doi.org/10.5061/dryad.gqnk98smc37.\n\nThis project contains the following underlying data:\n\nData file 1: (Demographic data of the participant at before the start of the Evening dose)\n\nData file 2: (Demographic data of the participant at before the start of the Morning dose)\n\nDryad: Pharmacodynamics parameters, https://doi.org/10.5061/dryad.k0p2ngf7b38.\n\nThis project contains the following underlying data:\n\nData file 1 (Cumulative amount of glucose excreted over the 24 hours at the Morning dose)\n\nData file 2 (Cumulative amount of glucose excreted over the 24 hours at the Evening dose)\n\nData file 3 (Pharmacodynamics evaluation Urinary glucose secretion)\n\nData file 4 (Pharmacodynamics parameters of Morning and Evening doses of Empagliflozin)\n\nDryad: Pharmacokinetics parameters, https://doi.org/10.5061/dryad.brv15dv8j39.\n\nThis project contains the following underlying data:\n\nData file 1 (Plasma concentration(s) levels of the 16 participants at the Evening dose)\n\nData file 2 (Plasma concentration(s) levels of the 16 participants at the Morning dose)\n\nData file 3 (Pharmacokinetics parameters of the 16 participants at the Evening dose)\n\nData file 4 (Pharmacokinetics parameters of the 16 participants at the Morning dose)\n\nData file 5 (AUC0-t and AUC0-inf of the 16 participants for both evening and morning doses).\n\nDryad: Bioanalytical method validation, https://doi.org/10.5061/dryad.brv15dv8j39.\n\nDryad: Protocol, case report(s), ethics approvals, inclusion and exclusion criteria, https://doi.org/10.5061/dryad.7h44j0zt140.\n\nThis project contains the following underlying data:\n\nData file 1: (The trial protocol)\n\nData file 2: (Empty form of case report) this file is not at https://doi.org/10.5061/dryad.7h44j0zt1\n\nData file 4: (Inclusion and Exclusion criteria for the enrollment of the participants in the trial)\n\nData file 5: (Ethics committee approval(s) of AinShms University)\n\nDryad: TREND checklist for ‘The effect of morning versus evening administration of empagliflozin on its pharmacokinetics and pharmacodynamics characteristics in healthy adults: a two-way crossover non-randomised trial’, https://doi.org/10.5061/dryad.gqnk98smc37.\n\nData is licensed under a CC0 1.0 Universal (CC0 1.0) Public Domain Dedication license.", "appendix": "Acknowledgements\n\nI thank Yulia Kovalenko, Misr International University for her assistance in revising the manuscript.\n\n\nReferences\n\nJaveed N, Matveyenko AV: Circadian Etiology of Type 2 Diabetes Mellitus. 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PubMed Abstract \| Publisher Full Text\n\nYoshioka H, Ohishi R, Hirose Y, et al.: Chronopharmacology of dapagliflozin-induced antihyperglycemic effects in C57BL/6J mice. Obes Res Clin Pract. 2019; 13(5): 505–10. PubMed Abstract \| Publisher Full Text\n\nJardiance. Diabetes.co.uk. 2019. Reference Source\n\nHeise T, Seewaldt-Becker E, Macha S, et al.: Safety, tolerability, pharmacokinetics and pharmacodynamics following 4 weeks’ treatment with empagliflozin once daily in patients with type 2 diabetes. Diabetes Obes Metab. 2013; 15(7): 613–621. PubMed Abstract \| Publisher Full Text\n\nHeise T, Seman L, Macha S, et al.: Safety, tolerability, pharmacokinetics, and pharmacodynamics of multiple rising doses of empagliflozin in patients with type 2 diabetes mellitus. Diabetes Ther. 2013; 4(2): 331–45. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSeman L, Macha S, Nehmiz G, et al.: Empagliflozin (BI 10773), a potent and selective SGLT2 inhibitor, induces dose-dependent glucosuria in healthy subjects. Clin Pharmacol Drug Dev. 2013; 2(2): 152–61. PubMed Abstract \| Publisher Full Text\n\nLaffel LMB, Tamborlane WV, Yver A, et al.: Pharmacokinetic and pharmacodynamic profile of the sodium-glucose co-transporter-2 inhibitor empagliflozin in young people with Type 2 diabetes: a randomized trial. Diabet Med. 2018; 35(8): 1096–104. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMacha S, Jungnik A, Hohl K, et al.: Effect of food on the pharmacokinetics of empagliflozin, a sodium glucose cotransporter 2 (SGLT2) inhibitor, and assessment of dose proportionality in healthy volunteers. Int J Clin Pharmacol Ther. 2013; 51(11): 873–9. PubMed Abstract \| Publisher Full Text\n\nElDash R: Demographic data. Dryad. 2021. http://www.doi.org/10.5061/dryad.gqnk98smc\n\nElDash R: Pharmacodynamics parameters. Dryad. Dataset, 2021. http://www.doi.org/10.5061/dryad.k0p2ngf7b\n\nElDash R: Pharmacokinetics parameters. Dryad. 2021. http://www.doi.org/10.5061/dryad.brv15dv8j\n\nElDash R: Protocol, case report(s), informed consent, inclusion and exclusion criteria. Dryad. 2021. http://www.doi.org/10.5061/dryad.7h44j0zt1" }	[ { "id": "84059", "date": "12 May 2021", "name": "Hesham Basma", "expertise": [ "Reviewer Expertise Molecular and cell biology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nHerein, Rana M. ElDash and colleagues report findings on the pharmacokinetics and pharmacodynamics characteristics of morning versus evening administration of empagliflozin in healthy adults. The authors concluded that there is no statistical significance between the morning and evening doses of empagliflozin. The findings are very timely and would be well received. However, there are few minor revisions that should be addressed by the authors.\nIn the abstract, methods section, please rephrase the following sentence to be clearer, “in the evening, and after a seven-day washout period, the morning”\n\nSeveral studies have been done for Statins chronotherapy. It is very relevant to the current study. I would encourage the authors to include a reference for it in the introduction.\n\nIn table one, the same individuals participated in both phases and it is redundant to mention the same numbers in phases one and two, I would suggest making it one column (with parameter in the header instead of phase1 and phase2).\n\nIs it possible to include Standard Error (S.E.) in figure 3?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "85636", "date": "28 May 2021", "name": "Ashraf Abdel-Naim", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe effect of morning versus evening administration of empagliflozin on its pharmacokinetics and pharmacodynamics characteristics in healthy adults: a two-way crossover, non-randomized trial.\nComment 1: the manuscript is well-written, and the study hypothesis is clear, although; more information about previous studies with similar ideas should be addressed in detail and specifically focusing on the number of patients enrolled on those similar studies\n\nComment 2: I believe the manuscript will benefit from limitation section that address all the concerns from the study\n\nComment 3: I find it really interesting that information in Table 1 are almost the same after a 7-day washout period. Readings such as fasting blood glucose, Triglycerides, HDL, LDL can be changed very dramatically in one day.\n\nMinor Comments:\nIn the abstract section: in the methods part, line 9 ‘was done prior to injection in LC/MS/MS’ please rephrase this sentence.\n\nIn the intervention section, line 13: please rephrase this sentence ‘in the evening or morning’.\n\nIn the intervention section, last line ‘dose was administered under close supervision of a medical investigator’ should be added with line 4.\n\nIn Endpoint section: ‘pharmacokinetic endpoint was to determine’ should be ‘pharmacokinetic endpoints were to determine’.\n\nIn discussion section: paragraph 3‘Our study examined the effect of the SGLT2i with empagliflozin, taking into account that the transport of solutes in and out of the kidney are controlled by the circadian clock. It has been shown that NH3 and SGLT1 are controlled by circadian oscillations proteins (Per 1 and BMA1), affecting the mRNA transcription’ need to be clearer.\n\nI suggest approval with minor revision.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "85394", "date": "28 May 2021", "name": "Samar Darwish", "expertise": [ "Reviewer Expertise pharmacokinetics", "pharmacodynamics", "endocrine disorders", "Drug discovery", "inflammatory diseases", "neurological disorders", "single-cell technologies." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this manuscript, the authors investigated the chronopharmacological effects of morning vs evening empagliflozin administration (10 mg) to healthy Egyptian adults, by studying the pharmacokinetics and some pharmacodynamics characters of the drug. The study sheds the light on the possibility that circadian rhythm can affect drug properties, and may inspire other researchers to study this theory in diabetic patients. However, there are few minor revisions that I advise the authors to take in concern:\nAbstract section:\nIn Methods part: please add “in” before “the morning” word to be more clear for the readers. In Methods part: please mention “The urine glucose excreted” before its abbreviation “UGE” and remove “glucose excreted” from the Results part. In Conclusion part: would you please mention the P value used for the statistical findings, for it will help other researchers for further studies.\n\nIntroduction section:\n\nPlease rephrase the sentence “the blood pressure lowering drug Fimasartan shows pronounced blood pressure lowering (readjusting the dipping pattern) at night compared to morning dose.” In the 9th paragraph to be more clear.\n\nMethods section:\nPlease also mention the number of participants in the text as you mentioned in the figure. In the Eligibility criteria part: you have mentioned that the study excluded some participants, while in the study design figure the excluded number was zero. Please check. In the Endpoints part: adjust the abbreviation meaning of UGT as requested before.\n\nResults section:\nIn table 1: Why did you express the data with median instead of the mean?? What is the difference between the data represented in figure 1 and figure 2? If there, please clarify. If applicable, I recommend mentioning the statistical significance or insignificance when comparing the results of evening dose to the morning dose all over the result section.\n\nDiscussion section:\nIn the first paragraph: the sentence starting with “The following examples show…..” is too long, please try to rephrase it. In the second paragraph: I’d prefer to replace the sentence “which may suggest that further studies to be done on the expression of SGLT1 and SGLT2 at the proximal tubules in the kidney (in which empagliflozin inhibit) are required” with the following sentence “which may suggest that further studies are required on the inhibitory site of empagliflozin, which is the expression of SGLT1 and SGLT2 at the proximal tubules” In the 3rd paragraph: clarify what did “i” refer to in the abbreviation “SGLT2i” Also in the 3rd paragraph: “Na+/H+ ex- changer 3” should be mentioned before its abbreviation “NH3”, and removed from the 4th paragraph It would be better to rephrase paragraph number 5.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-321
https://f1000research.com/articles/9-1489/v1	18 Dec 20	{ "type": "Systematic Review", "title": "Incidence of acute pulmonary embolism among patients hospitalized with COVID-19: a systematic review and meta-analysis", "authors": [ "Mohamed S. Munner", "Charles A. Ritchie", "Ibrahim H. Elkhidir", "Doaa T. Mohammadat", "Hussein J. Ahmed", "Khalid A. Altayeb", "Rawan Z. Yassin", "Riyan M. Hassan", "Saada A. Hamad", "Mohammed Nimir", "Osman S. Hamid", "Margaret M. Johnson", "Tathagat Narula", "Young Erben", "Rabih G. Tawk", "David A. Miller", "Vivek Gupta", "Zlatko Devcic", "William D. Freeman", "Beau B. Toskich", "Mohamed S. Munner", "Charles A. Ritchie", "Ibrahim H. Elkhidir", "Doaa T. Mohammadat", "Khalid A. Altayeb", "Rawan Z. Yassin", "Riyan M. Hassan", "Saada A. Hamad", "Mohammed Nimir", "Osman S. Hamid", "Margaret M. Johnson", "Tathagat Narula", "Young Erben", "Rabih G. Tawk", "David A. Miller", "Vivek Gupta", "Zlatko Devcic", "William D. Freeman", "Beau B. Toskich" ], "abstract": "Background: Coronavirus disease 2019 (COVID-19) is a global pandemic, which is associated with venous thromboembolism and pulmonary embolism (PE). This study aimed to estimate the pooled incidence of PE among patients hospitalized with COVID-19 within the published literature. Methods: This systematic review and meta-analysis was performed according to PRISMA guidelines. An electronic search using MEDLINE /PubMed, ScienceDirect, Cochrane, and OpenGray databases was conducted May 19th, 2020. Search terms included \"COVID 19\", \"SARS-CoV-2”, \"coronavirus disease 2019\", \"2019-nCoV\", \"Wuhan coronavirus\", “Pulmonary embolism”, \"pulmonary thromboembolism\", “Pulmonary embol”, “pulmonary thrombo” and “PE”. Eligible studies included sufficient data to calculate the incidence of PE diagnosed during hospitalization in patients with COVID-19. Case reports were excluded. Quality was assessed using the Newcastle-Ottawa scale (observational cohort and case-control), AXIS tool (cross-sectional), and quality assessment tool (case series). Demographics and PE incidence data were extracted from the included studies and analyzed with R language. The pooled incidence of PE in patients hospitalized with COVID-19 was calculated. Results: The database search identified 128 records. Ten observational studies were eligible and were included in the meta-analysis with a total of 1722 patients (mean age= 63.36). The pooled PE incidence in patients hospitalized with COVID-19 was 17% (95% CI: 0.1-0.26). There was a high degree of study heterogeneity (I2 = 94%, p<0.01). Conclusion: The pooled PE incidence in patients hospitalized with COVID-19 is 17%. This increased incidence is greater than that previously reported in the general population of non-COVID-19. Attention and further investigation of this risk is warranted.", "keywords": [ "Pulmonary Embolism", "Coronavirus", "COVID-19", "Venous thromboembolism", "Incidence", "Meta-analysis" ], "content": "Introduction\n\nIn December 2019, pneumonia of unknown cause was detected in Wuhan, China1. The causative agent was identified and named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)2. On March 11th, 2020, the World Health Organization characterized the coronavirus disease of 2019 (COVID-19) as a pandemic1,3, resulting in 53,507,282 and 1,305,164 COVID-19-related cases and deaths, respectively, as of November 15th, 20204. While COVID-19 is primarily a pulmonary disease, there are multiple other pathologic manifestations and complications, including pulmonary embolism (PE)5.\n\nThe relationship between COVID-19 and thromboembolism is becoming established in the literature5. Thromboembolism has been previously associated with zoonotic coronaviruses6 and may be attributed to several factors including; a hypercoagulable state associated with severe infection or inflammation7, COVID-19 associated hemostatic abnormalities8,9, recumbence7,10–12, and possible drug interactions between investigational COVID-19 therapies (Lopinavir/ritonavir) and antithrombotics5.\n\nThis systematic review and meta-analysis analyzed and estimated the pooled PE incidence from published literature of patients hospitalized with COVID-19 who developed PE.\n\n\nMethods\n\nThis meta-analysis was performed following the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) reporting guideline13,14. Institutional review board approval was not required for this study.\n\nA systematic literature search was performed on May 19th, 2020 using Medline/PubMed, Cochrane, OpenGrey, and ScienceDirect. PubMed, Cochrane, and OpenGrey were queried for the following strategy: (\"COVID 19\" OR \"SARS-CoV-2” OR \"coronavirus disease 2019\" OR \"2019-nCoV\" OR \"Wuhan coronavirus\") AND (“Pulmonary embolism” OR \"pulmonary thromboembolism\" OR “Pulmonary embol” OR “pulmonary thrombo” OR “PE”). ScienceDirect was queried using the same variables only without asterisks.\n\nDuplicate citations and older versions of the same study population were removed. All included study bibliographies were screened for additional articles investigating PE in patients hospitalized with COVID-19. All eligible studies underwent full-text screening.\n\nStudies reporting data sufficient to estimate PE incidence of patients hospitalized with COVID-19 were included in the analysis. Only English and published studies were included. Studies with insufficient data, case reports, editorials, proposals, and abstracts without full-text were excluded. The title and abstract of retrieved articles were screened by two independent reviewers for potential inclusion. Any discrepancy between the reviewers was resolved by consensus with two additional reviewers.\n\nAppraisal of individual study quality was performed by two independent reviewers using the Newcastle–Ottawa scale, AXIS tool, and quality assessment tool for observational cohort and case-control, cross-sectional, and case series, respectively15–17. Articles were deemed of good quality if they scored 50% or more using an arbitrary cut-off.\n\nInformation was sought from each included study are: characteristics of participants (including age, gender, BMI, D dimer of patient with COVID-19,, and total patients in the study), summary about included studies (first author, country, year of publication, study period, study design, method of diagnosing COVID-19) and the main data for meta-analysis (total patients in each study i.e., sample size and number of patients developing PE from the total). The data was subsequently extracted by five independent reviewers utilizing Microsoft Excel® 2016 (Microsoft Corporation, Redmond, WA).\n\nDue to skewed proportions, the random effect model was used to pool the individual estimates through LOGIT transformation instead of double arcsine transformation to avoid a paradoxical effect upon back-transformation18,19.\n\nStatistical analysis was performed using R language v.420, using “meta” and ”metafor” packages21–23. Random effects models were utilized to accommodate for the heterogeneity in the reported pooled incidences. Statistical heterogeneity was estimated using I2 statistics and further assessed using subgroup analysis, meta-regression, influence analysis, and Gosh analysis. Publication bias was evaluated by both the Egger test and funnel plot visual analysis.\n\n\nResults\n\nThe search yielded 81 and 47 records in both Medline/PubMed and ScienceDirect, respectively. No records were identified in OpenGrey and Cochrane databases. After eliminating duplicate data, 125 studies were included for abstract screening, of which 76 were excluded due to insufficient data. Full-text screening of the remaining 49 studies excluded 38 records with an agreement kappa of 0.813. Of the remaining studies, one scored <50% on the quality assessment tool, leaving ten studies for pooled analysis (Table 1). Nine studies were from Europe and one study was from North America (Table 1). Studies were divided into descriptive and analytic categories for subgroup analysis (Table 1). The reported PE incidence among patients hospitalized with COVID-19 in all included studies ranged from 3–35% (Table 2). Details of the selection process are summarized in (Figure 1).\n\nPE: pulmonary embolism; CTPA: CT pulmonary angiography.\n\nPatients had a mean age of 63 years. The incidence of PE was noted to be higher in males (Table 3). The D-dimer levels were specified between PE group and non-PE group in only three studies, while the remaining either reported it improperly or had missing data (Table 3).\n\n*data in non-PE group\n\nThough anatomical distribution of PE was mentioned in most studies (except Lorant et al. and Grillet et al.)29,30, the clinical classification of PE into massive and sub-massive was not mentioned.\n\nThe pooled incidence of PE in hospitalized patients with COVID-19 was 17% (95% CI: 11-26%) (Figure 2). I2 test revealed significant study heterogeneity (I2= 94%, p<0.01). The risk of publication bias was tested for by Egger test (t-value = -1.867, p=0.0989) and examined visually using a funnel plot (Figure 3).\n\nThe pooled PE incidence was estimated at 11% (95% CI: 5-23%) and 27% (95% CI 22-34%) in the analytic and descriptive studies, respectively. Higher heterogeneity was detected among the analytic group (I2= 95%, p<0.01), compared to the descriptive group (I2= 48%, p=0.05) (Figure 4).\n\nWhen categorizing the included studies into analytic or descriptive, the meta-regression model showed that the study design was significantly associated with the difference in PE incidence (p<0.05) (Figure 5).\n\n\nDiscussion\n\nPE is the most common thromboembolic complication occurring in patients with COVID-1924,30,32. This systematic review and meta-analysis estimated a pooled PE incidence among patients hospitalized with COVID-19 at 17% (95% CI: 10-26%) (Figure 2)24–33. This pooled PE incidence is higher than the PE incidence of the general population, and most importantly, higher than that of hospitalized patients with other medical conditions34–40.\n\nThis reported PE incidence could represent an over or under estimation of the true incidence. The increasing knowledge of higher incidences of venous thromboembolism in patients with COVID-19 may have led to a selection bias due to lower threshold of CT pulmonary angiography (CTPA) utilization41. An underestimation is known to occur in the general population for the diagnosis of PE related to the majority of massive PE being diagnosed on post-mortem examination. Similarly, patients with COVID-19 could be assumed to have a lower reported incidence of PE diagnosis due to; limited CT scan availability, patients instability, concern over hospital exposure to others related to transportation, early hospital mortality and death outside of healthcare5,42–44.\n\nMost of the patients included in this meta-analysis were males and aged sixty years and above. Not only was PE higher in elderly and males patients hospitalized with COVID-19, but also other COVID-19 related complications and death were seen more in this population45,46. This observation is consistent with the higher PE incidence across the elderly in the general population as well47. Therefore, hospitalized male and elderly patients with COVID-19 could be at a higher risk of developing PE compared to their COVID-19-afflicted female and young counterparts, respectively.\n\nTo account for the variability of CTPA timing effect on PE incidence, a study assigned each patient with COVID-19 two time points in the CTPA and demonstrated a higher incidence at the later time point27. Such an observation may help in interpreting the variation in PE incidence across different studies with similar population characteristics and may also highlight underestimation of PE incidence in population with single time CTPA.\n\nPE mortality was reported in only 3 out of the 10 studies using different formats24,25,27,28,33. Therefore, the estimated pooled mortality could not be calculated.\n\nThis study was designed and executed in accordance with PRISMA guidelines. The heterogonous severity of the included COVID-19 cohort included the complete spectrum of hospitalized patients with COVID-19. Therefore, this estimated incidence could apply to any hospitalized patients with COVID-19, regardless of disease acuity. However, these findings need to be interpreted in the context of some limitations. First, neither articles in non-indexed journals nor non-published papers were searched, which may have introduced some publication bias. Second, the inclusion of studies published only in English literature may have led to language bias. Third, the presence of a subpopulation in whom PE was suspected and CTPA could not be performed for various reasons, such as allergy to contrast material, and could result in skewed incidence. Fourth, both clinical indications for CTPA and PE classification or risk stratification were not specified in the different studies. Sixth, due to variability of the way of reporting specific data on some variables (d-dimer, mortality data), it was difficult to make meaningful predictions. Lastly, some relevant clinical and para-clinical variables were not mentioned.\n\nFurther research is warranted to accurately characterize the patients hospitalized with COVID-19 who develop PE in terms of; risk factors profile, PE diagnostic indications, CTPA timing, PE prophylaxis and management, and PE pathogenesis.\n\n\nConclusion\n\nThis systematic review and meta-analysis reported a pooled PE incidence among patients hospitalized with COVID-19 at 17%, suggesting that almost in every five hospitalized patients with COVID-19, one may develop PE. This represents around a 243-fold increase in incidence when compared to the general population. Healthcare professionals should be aware of this observed increase in risk of PE incidence among patients hospitalized with COVID-19. Development of accurate and precise risk stratification scores and point of care biomarkers to guide prophylactic and therapeutic strategies in this vulnerable population are warranted.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nOpen Science Framework: Codes and PRISMA checklist, https://doi.org/10.6084/m9.figshare.13347473.v248.\n\nThis project contains the following extended data:\n\n- Code for publishing plots (.R file).\n\n- Code for meta-analysis (.R file).\n\nOpen Science Framework: PRISMA checklist for ‘Incidence of acute pulmonary embolism among patients hospitalized with COVID-19: a systematic review and meta-analysis’, https://doi.org/10.6084/m9.figshare.13347473.v248.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nWeb1-WHO: Coronavirus (COVID-19) events as they happen. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMiddeldorp S, Coppens M, van Haaps TF, et al.: Incidence of venous thromboembolism in hospitalized patients with COVID-19. J Thromb Haemost. 2020; 18(8): 1995–2002. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHelms J, Tacquard C, Severac F, et al.: High risk of thrombosis in patients with severe SARS-CoV-2 infection: a multicenter prospective cohort study. Intensive Care Med. 2020; 46(6): 1089–98. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBompard F, Monnier H, Saab I, et al.: Pulmonary embolism in patients with Covid-19 pneumonia. Eur Respir J. 2020; 56(1): 2001365. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPoyiadi N, Cormier P, Patel PY, et al.: Acute Pulmonary Embolism and COVID-19. Radiology. 2020; 297(3): E335–E338. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGrillet F, Behr J, Calame P, et al.: Acute Pulmonary Embolism Associated with COVID-19 Pneumonia Detected by Pulmonary CT Angiography. Radiology. 2020; 296(3): E186–E188. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLeonard-Lorant I, Delabranche X, Severac F, et al.: Acute Pulmonary Embolism in COVID-19 Patients on CT Angiography and Relationship to D-Dimer Levels. Radiology. 2020; 296(3): E189–E191. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPoissy J, Goutay J, Caplan M, et al.: Pulmonary Embolism in COVID-19 Patients: Awareness of an Increased Prevalence. Circulation. 2020; 142(2): 184–186. PubMed Abstract \| Publisher Full Text\n\nKlok FA, Kruip MJHA, van der Meer NJM, et al.: Confirmation of the high cumulative incidence of thrombotic complications in critically ill ICU patients with COVID-19: An updated analysis. Thromb Res. 2020; 191: 148–50. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLlitjos JF, Leclerc M, Chochois C, et al.: High incidence of venous thromboembolic events in anticoagulated severe COVID-19 patients. J Thromb Haemost. 2020; 18(7): 1743–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWiener RS, Schwartz LM, Woloshin S: Time trends in pulmonary embolism in the United States: Evidence of overdiagnosis. Arch Intern Med. 2011; 171(9): 831–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOger E: Incidence of venous thromboembolism: a community-based study in Western France. EPI-GETBP Study Group. Groupe d’Etude de la Thrombose de Bretagne Occidentale. Thromb Haemost. 2000; 83(5): 657–60. PubMed Abstract \| Publisher Full Text\n\nCook D, Crowther M, Meade M, et al.: Deep venous thrombosis in medical-surgical critically ill patients: Prevalence, incidence, and risk factors. Crit Care Med. 2005; 33(7): 1565–71. 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PubMed Abstract \| Publisher Full Text\n\nvan Dam LF, Kroft LJM, van der Wal LI, et al.: Clinical and computed tomography characteristics of COVID-19 associated acute pulmonary embolism: A different phenotype of thrombotic disease? Thromb Res. 2020; 193: 86–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCarsana L, Sonzogni A, Nasr A, et al.: Pulmonary post-mortem findings in a series of COVID-19 cases from northern Italy: a two-centre descriptive study. Lancet Infect Dis. 2020; 20(10): 1135–1140. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKarwinski B, Svendsen E: Comparison of clinical and postmortem diagnosis of pulmonary embolism. J Clin Pathol. 1989; 42(2): 135–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSweet PH, Armstrong T, Chen J, et al.: Fatal pulmonary embolism update: 10 years of autopsy experience at an academic medical center. JRSM Short Rep. 2013; 4(9): 204253331348982. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWu C, Chen X, Cai Y, et al.: Risk Factors Associated with Acute Respiratory Distress Syndrome and Death in Patients with Coronavirus Disease 2019 Pneumonia in Wuhan, China. JAMA Intern Med. 2020; 180(7): 934–943. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRichardson S, Hirsch JS, Narasimhan M, et al.: Presenting Characteristics, Comorbidities, and Outcomes Among 5700 Patients Hospitalized With COVID-19 in the New York City Area. JAMA. 2020; 323(20): 2052. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHippisley-Cox J, Coupland C: Development and validation of risk prediction algorithm (QThrombosis) to estimate future risk of venous thromboembolism: Prospective cohort study. BMJ. 2011; 343: d4656. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nElkhidir I: Codes and PRISMA checklist. figshare. Software. 2020. http://www.doi.org/10.6084/m9.figshare.13347473.v2" }	[ { "id": "77814", "date": "10 Mar 2021", "name": "Xingshun Qi", "expertise": [ "Reviewer Expertise Systematic review and meta-analysis", "Clinical studies" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper is too simple, especially the Results section focusing on the pooled incidence of PE. Data analysis should be expanded to explore the risk factors and impact of PE. Can the relationship between PE and COVID-19 severity be established? Additionally, it is suggested that several subgroups can be used to analyze the source of heterogeneity.\nSome published papers regarding a similar topic should be included. The Results section of the Abstract could introduce more contents regarding the main results of the study, while the Methods section can be shortened.\nThe Introduction section says, “In December 2019, pneumonia of unknown cause was detected in Wuhan, China”. Such words are not appropriate; the first case of COVID-19 is still unclear.\nIt is more appropriate to use MOOSE guidelines for a systematic review and meta-analysis of articles discussing the incidence.\nCurrent research is not registered on PROSPERO.\nThe authors say, “A systematic literature search was performed on May 19th, 2020”. This paper is a bit older. It should be updated and collect more new data.\nThe data analysis is a bit simple and the significance of the p value was not defined.\nThe research flow chart is not concise enough. What is the meaning of \"36 records with no sufficient\"?\nThe authors said \"Random effects models were utilized to accommodate for the heterogeneity in the reported pooled incidences\". However, they still introduced fixed-effect models in figures.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? No\n\nIs the statistical analysis and its interpretation appropriate? Partly\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [ { "c_id": "6554", "date": "26 Apr 2021", "name": "Hussein Ahmed", "role": "Author Response", "response": "Dear reviewer, Thank you for your valuable comments, we did a systematic review and meta-analysis regarding the incidence of PE among patients hospitalized due to COVID-19. Our ultimate aim is to calculate the pooled incidence of PE to quantify the importance of that deadly condition. when we did this review, it was the first review at that time, and so published papers regarding a similar topic were not included. The Introduction section we say, “In December 2019, pneumonia of unknown cause was detected in Wuhan, China”. By this we mean as the WHO reported the first appearance of COVID-19 in China and not the first case of COVID-19. It is an advantage but not necessary to register our research at PROSPERO. We have also corrected the missing word in the flowchart to be more concise and we removed the fixed effect model from the figures." } ] } ]	1	https://f1000research.com/articles/9-1489
https://f1000research.com/articles/10-320/v1	26 Apr 21	{ "type": "Opinion Article", "title": "Bioimage analysis workflows: community resources to navigate through a complex ecosystem", "authors": [ "Perrine Paul-Gilloteaux", "Sébastien Tosi", "Jean-Karim Hériché", "Alban Gaignard", "Hervé Ménager", "Raphaël Marée", "Volker Baecker", "Anna Klemm", "Matúš Kalaš", "Chong Zhang", "Kota Miura", "Julien Colombelli", "Sébastien Tosi", "Jean-Karim Hériché", "Alban Gaignard", "Hervé Ménager", "Raphaël Marée", "Volker Baecker", "Anna Klemm", "Matúš Kalaš", "Chong Zhang", "Kota Miura", "Julien Colombelli" ], "abstract": "Workflows are the keystone of bioimage analysis, and the NEUBIAS (Network of European BioImage AnalystS) community is trying to gather the actors of this field and organize the information around them. One of its most recent outputs is the opening of the F1000Research NEUBIAS gateway, whose main objective is to offer a channel of publication for bioimage analysis workflows and associated resources. In this paper we want to express some personal opinions and recommendations related to finding, handling and developing bioimage analysis workflows. The emergence of \"big data” in bioimaging and resource-intensive analysis algorithms make local data storage and computing solutions a limiting factor. At the same time, the need for data sharing with collaborators and a general shift towards remote work, have created new challenges and avenues for the execution and sharing of bioimage analysis workflows. These challenges are to reproducibly run workflows in remote environments, in particular when their components come from different software packages, but also to document them and link their parameters and results by following the FAIR principles (Findable, Accessible, Interoperable, Reusable) to foster open and reproducible science. In this opinion paper, we focus on giving some directions to the reader to tackle these challenges and navigate through this complex ecosystem, in order to find and use workflows, and to compare workflows addressing the same problem. We also discuss tools to run workflows in the cloud and on High Performance Computing resources, and suggest ways to make these workflows FAIR.", "keywords": [ "Bioimage analysis", "workflows", "components", "collections", "FAIR principles", "NEUBIAS", "remote computing", "scientific workflow management systems", "knowledge database" ], "content": "Introduction\n\nWorkflows are the keystone of bioimage analysis,1,2 and the NEUBIAS community is trying to gather the actors of this field and organize the information around them. One of its most recent outputs is the opening of the F1000Research NEUBIAS gateway, whose main objective is to offer a channel of publication for bioimage analysis workflows3 and associated resources.\n\nIn this paper, we aim to express some personal opinions and recommendations related to finding, handling and developing bioimage analysis workflows.\n\nA bioimage analysis workflow is defined as a set of computational tools assembled in a specific order to process bioimages and estimate some parameters relevant to the biological system under study. To classify these computational tools, in the NEUBIAS community, we have defined these terms: workflows, components and collections1,4 as follows. A workflow is built as a sequence of components coming from one or multiple software packages. It takes an image as input and outputs processed images, numerical values and/or annotations (e.g. biological objects outlines). A component is the software implementation of an image processing or analysis algorithm. We call collection the software package that gathers components and can be in the form of a generalist software platform such as ImageJ and Fiji,5 Icy,6 CellProfiler:7 specialized platforms, such as analyzing a specific modality of microscopy e.g. super resolution image data;8 or computationally optimized libraries such as ImgLib29 or ITK.10 Most of the time, standalone components cannot solve complex bioimage analysis problems on their own – that is why they need to be carefully assembled.\n\nThe emergence of resource-intensive analysis algorithms, e.g. supervised machine learning with convolutional neural networks, and of \"big data” in bioimaging make local data storage and computing solutions a limiting factor. At the same time, the need for data sharing with collaborators and a general shift towards remote work, have created new challenges and avenues for the execution and sharing of bioimage analysis workflows.\n\nThese challenges are to reproducibly run workflows in remote environments, in particular when their components come from different collections, but also to document them and link their parameters and results by following the FAIR principles (Findable, Accessible, Interoperable, Reusable)11 to foster open and reproducible science.\n\nIn this opinion paper we focus on giving some directions to the reader to tackle these challenges and navigate through this complex ecosystem, in order to find and use workflows (and components), and to compare workflows addressing the same problem. We also discuss tools to run workflows in the cloud and on High Performance Computing (HPC) resources, and suggest ways to make these workflows FAIR.\n\n\nFinding workflows or components for a specific biological problem or task\n\nThe first challenge in the creation of a workflow is to avoid duplicating the effort and being able to easily find and customize a workflow that has been used for a similar biological problem. Today, browsing the documentation of bioimage analysis tools, or asking a specific question in a generic forum, such as the newly created Image.sc forum,12 will help guide the biologist or microscopist to existing tools. We believe that while this can be a good starting point it may not be sufficient. The NEUBIAS training courses13,14 and the NEUBIAS Academy (see15 in this Gateway) are two of the educational resources that can also help finding and adapting existing workflows. Exposing tools and workflows in a knowledge database has also been identified as very useful by the community. Table 1 illustrates some examples of such databases where bioimage analysts can reference their workflows using the proposed standardized framework and vocabulary in order to make them findable.\n\nSome examples of such databases where bioimage analysts can reference their workflows.\n\nBIII, the BioImage Informatics Index, has been created in the context of the NEUBIAS network and with the effort of tens of volunteers. Software tools (>1343), image databases for benchmarking (>24) and training materials (>71) for bioimage analysis are referenced and curated following standards constructed by the community. The range of software tools available includes workflows (>172), specific components (>898), and collections (>302). All entries are exposed following FAIR principles and accessible for other usage. They are described using Edam Bio Imaging,16 a dedicated extension of the generalist EDAM ontology17 for bioimage analysis, bioimage informatics, and bioimaging. It is developed in a community spirit, in collaboration between numerous bioimaging experts and ontology developers. It is used in BIII to describe the applications of these tools, by describing the operations performed (such as segmentation, visualization, or lower level operation) and the field of applications of these tools such as the imaging modalities to which it can be applied (i.e. EDAM Bioimaging Ontology). EDAM Bioimaging has now a solid basis. This basis is incrementally defined at specific meetings (i.e. taggathons) where suggestions for new terms, crowd-sourced from free tags by BIII users, are inspected and moderated for inclusion, or contrasted by bioimage analysis experts when no term is found adequate.\n\nSimilar initiatives exist, either for a broader range of applications, for example bio.tools,18,19 which has gathered more than 20000 software tools in the full range of life science applications, or for more specific application topics, for example Quantitative Plant, which focusses on tools for the analysis of image data of plants20 or BioImage.io for pre trained AI deep learning models.\n\nBy feeding the description of a workflow in the knowledge database BIII (following the recommendation provided), and thanks to workflow/tools interoperability standards, these workflows can be found by other bioimage analysts or automatically discovered and consumed by other registries, such as bio.tools, to reach a broader community.\n\n\nComparing workflows\n\nOnce a candidate workflow has been found, the natural question is then if it is the best solution for the particular task one wants to solve. Table 2 shows three examples of resources comparing workflows.\n\nBIAFLOWS21 is an open-source web platform to reproducibly deploy and publicly benchmark image analysis workflows with a strong focus on microscopy bioimages. The database stores scientific datasets, metadata, and versioned image analysis workflows with parameters optimized for the corresponding datasets. The workflows can be run remotely. The results (e.g. object annotations) from different workflows (or from runs with different parameter values) can be visualized remotely as an overlay on the original images. When the images hold reference annotations, the results are automatically benchmarked by commonly adopted benchmark metrics targeting one of the nine currently supported problem classes. The benchmark metrics of each workflow run can be browsed per image or as overall statistics over whole datasets. BIAFLOWS brings an automated mechanism leveraging DockerHub to encapsulate, version and make the workflows and their complete execution environment available upon every new release. Overall, BIAFLOWS enables integration and web-based evaluation of heterogeneous workflows originally written for diverse languages and libraries.\n\nThe Grand Challenge is a website cataloguing a set of challenges, focusing mostly on medical imaging. These challenges are usually hosted by a conference such as IEEE ISBI and run as an annual edition with specific reporting22,23 and they gather and evaluate competing workflows to solve a common bioimage analysis task. In the microscopy imaging communities, a particular effort has gone towards nuclei segmentation with the goal of developing a universal nuclei segmenter that works across different imaging modalities, as for instance with the Kaggle Data Science Bowl of 2018, providing a considerable amount of annotated data.24\n\n\nTowards reproducibility and interoperability in bioimage analysis\n\nThe current paradigm for bioimage analysts is to create workflows using a single platform or application, aka collection, for example Fiji,25 CellProfiler,7 or Icy.6\n\nBy allowing the possibility to script a workflow calling their components with simplified programming language, these platforms offer ways to share and document the workflows for other users. Besides script creation, there are also options to create sharable elements with no programming skills, as detailed in.26 This only requires the deployment of the software package to be run.\n\nThis reliance on graphical user interfaces favors the development of components built for a single collection. While this has stimulated the gathering of active communities around these collections, the coexistence of many multifunctional collections that are developed independently is not ideal for cloud deployment and FAIR principles. The graphical user interfaces are often not compatible with the type of remote computing offered by cloud technologies and the large collections contain largely overlapping components that are however not interoperable with each other. These collections therefore do not offer a unified and granular way of describing an image processing workflow. This situation also often requires users to learn multiple platforms to be able to complete their workflows. Code notebooks such as CodeOcean capsules or Jupyter notebooks offers also an easy access to cloud computing or HPC, but several aspect of workflow management are also left to the user, in particular data provenance.\n\nAt the same time that the field is shifting to running workflows in the cloud or high performance computing environments, there also comes the need to run more complex workflows integrating tools and data coming from different life science fields, such as genomics or proteomics data, or spatial transcriptomics. In addition to the integration of component from different communities, one can face the challenge to run again a previously created workflow and encounter versioning problems, with time and evolution of software packages and component versions. Specific configuration issues also make tedious the portability for the execution of a workflow from an environment to another, such as moving between HPCs or cloud computing platforms. While the use of virtual machines accessible from a web browser to emulate a personal desktop experience may be seductive, the bioimage analysis community should not isolate itself from other communities, and in particular not from bioinformatics community. Several bioinformatics communities have already started to tackle these issues through the use of scientific workflow management systems (SWMS)27,28 and standardized software packaging practices.29 These SWMS have also the advantage to tackle standardized workflow description, machine-readable as well as human readable, for FAIR principles. In comparison, the usual documentation, provided when documenting a workflow in bio image analysis current practices, is usually addressed to humans (which is already laudable and not yet common practice).\n\nOne of the key elements to enable reproducibility and portability is containerization and software packaging that facilitates the reliable creation of containers. Containerization consists in embedding a piece of software, and all its dependencies and specific configuration in one file called a container image, so that the software can run consistently across different computing environments. Table 3 shows examples of workflow management systems with usage in bioimage analysis. This containerization can be performed at the level of each individual workflow component (such as in Galaxy30,31), or for complete workflows (such as in BIAFLOWS21 or coming to grand-challenges). Biocontainers32 is proposing a standard and recipes for these containerizations, as well as a marketplace for the containers, today mostly for –omics data processing.\n\nAs a community we need to join this effort for a better exploitation and reproducibility by other communities of the imaging data produced by our workflows. One of the particularities of workflows in bioimage analysis is the need for visual and accurate feedback at critical workflow steps. This human-in-the-loop requirement has so far prevented the community from using SWMS more widely. But this is now changing as image processing tools and visual feedback are now getting incorporated in SWMS.21,31,33\n\n\nTowards findability and accessibility of image analysis workflows\n\nAt a general level in life-science and not specifically for the bioimage analysis community, coordination efforts are ongoing in the direction of the “FAIRification” of workflows, but also the ease to access HPC resources to run them. They are led by European Research Infrastructures, such as ELIXIR.34 ELIXIR is an intergovernmental organization that aims to coordinate the resources offered nationally for databases, software tools and access to cloud storage and HPCs, and associated training material. BIII, the finder tool mentioned above, is now for example part of the recommended interoperability resources. EOSC-Life is an ESFRI cluster project involving the 13 biomedical research infrastructures whose goal is to create an open, digital and collaborative space for biological and medical research in the European Open Science Cloud. This includes making image data and image processing and analysis workflows compliant with the FAIR principles, while enabling interoperability with tools and data from other life science domains as mandated by the European Commission. Galaxy30 has been identified and selected as an aggregator of communities, and selected by EOSC-Life as an exemplary workflow management system that promotes cross-communities interoperability in the cloud. This does not mean that the bioimage analysis community needs to restrict to this particular choice, but it means that the workflows have to be compatible with this choice and to prepare for a future where local compute resources will not anymore be used to run a workflow.\n\nTo ease this interoperability, a common description needs to be defined, in order to be able to make workflows interoperable and compatible with different infrastructure environments. The description of a workflow is different from the workflow itself: it is a human- and machine-readable description following standard syntax or vocabularies that will allow this workflow to be FAIR.35 A workflow should be associated with a standardized description (such as a unique identifier for the workflow itself, their component, but also their creator) and a description of its constitutive components and their configuration. The researchers who created the workflow can be identified by their ORCIDs. The Common Workflow Language36 could be used as a standard to describe workflows in an interoperable way since it has reached a sufficient level of maturation and flexibility. To further facilitate their findability by web search and indexing engines, lightweight metadata can be provided through the Schema.org controlled vocabularies or Bioschemas, a specific extension for Life Science resources.\n\nGalaxy is one of many SWMS, a more exhaustive list curated by the Common Workflow Language organization can be found here. Table 3 focuses on SWMS used in the bioimage analysis field, and it details their specificities. These specificities tend to support the message that the effort should not be in trying to push the implementation of workflows in only one solution, but rather to allow and ease the portability of workflows in multiple frameworks and execution environments, an approach supported by initiatives CWL. We then argue that these standards are key to facilitate the workflow ecosystems and further promote open and reproducible sciences.\n\n\nConclusion\n\nThe field of bioimage analysis, partly thanks to the NEUBIAS community, has been recently consolidated. Its community has contributed to the emergence of new tools to find, launch, compare and learn how to use and customize image analysis workflows. We believe that today the field has become mature enough to contribute to the general open science effort in life science and to enable better access to data and computational resources. This effort should help promote workflow sharing and reuse and a wider data integration and interoperability. We deeply encourage the bioimage analyst community, and by extension the associated software developer community, to sustain this effort and to rely on these tools. In particular, we encourage bioimage analysts to describe their workflows thoroughly by following the CWL standard, index them in BIII, and share them in SWMS such as BIAFLOWS compatible with Galaxy.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "Acknowledgements\n\nThis publication was supported by COST (European Cooperation in Science and Technology) under COST Action NEUBIAS (CA15124). The authors wish to thank all contributors for their continuous and invaluable input to the aforementioned online community resources developed under the framework of NEUBIAS (BIII, BIAFLOWS) and whose contributions are described and acknowledged in their respective publications.\n\n\nReferences\n\nMiura K, Paul-Gilloteaux P, Tosi S, et al.: Workflows and Components of Bioimage Analysis. In: Bioimage Data Analysis Workflows. Miura K, Sladoje N Éd. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nda Veiga Leprevost F, et al.: BioContainers: an open-source and community-driven framework for software standardization. Bioinformatics. août 2017; vol. 33, no 16, p. 2580–2582. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDietz C, et al.: Integration of the ImageJ Ecosystem in KNIME Analytics Platform. Front. Comput. Sci. mars 2020; vol. 2, p. 8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHarrow J, et al.: ELIXIR-EXCELERATE: establishing Europe’s data infrastructure for the life science research of the future. EMBO J. mars 2021; vol. 40, no 6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGoble C, et al.: FAIR Computational Workflows. Data Intell. janv. 2020; vol. 2, no 1-2, p. 108–121. Publisher Full Text\n\nAmstutz P, et al.: Common Workflow Language, v1.0. figshare. 2016; p. 5921760 Bytes. Publisher Full Text\n\nHarshil P, Karishma M, Angelova M, et al.: nf-core/imcyto: nf-core/imcyto v1.0.0 - Platinum Panda. Zenodo. 2020.\n\nvan Maldegem F, et al.: Characterisation of tumour immune microenvironment remodelling following oncogene inhibition in preclinical studies using an optimised imaging mass cytometry workflow. Cancer Biology, preprint, févr. 2021. Publisher Full Text\n\nEwels PA, et al.: The nf-core framework for community-curated bioinformatics pipelines. Nat. Biotechnol. mars 2020; vol. 38, no 3, p. 276–278. PubMed Abstract \| Publisher Full Text\n\nSchmied C, Steinbach P, Pietzsch T, et al.: An automated workflow for parallel processing of large multiview SPIM recordings. Bioinformatics. avr. 2016; vol. 32, no 7, p. 1112–1114. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMölder F, et al.: Sustainable data analysis with Snakemake. F1000Res. janv. 2021; vol. 10, p. 33. 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Publisher Full Text" }	[ { "id": "83885", "date": "20 May 2021", "name": "Katy Wolstencroft", "expertise": [ "Reviewer Expertise Scientific workflows", "FAIR data", "semantic data integration." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article describes current progress in the area of creating and sharing scientific workflows for bioimage analysis. The article describes community activities that allow the collection and comparison of workflows. It also discusses the necessity for describing workflows and their components in standardised ways, using common vocabularies.\nThe article serves as a gateway to bioimaging analysis workflow resources. As such, it is a good introduction and provides a clear overview. However, the reality of creating bioimaging analysis workflows, and particularly of reusing them, has some large bottlenecks. It would be beneficial to discuss, even if only briefly, what the bottlenecks are and describe current major challenges. The authors already mention interoperability and provenance as large challenges, but the connection to the data is another large challenge. Are datasets described in common formats? Are there existing ways to work with data generated with different proprietary formats? For this point, I miss a reference to the work of the Open Microscopy Environment community (e.g. 1).\n\nAre there common representations for bioimaging data? How practical is it to move such large datasets around for the purposes of analysis? For widespread use of bioimaging workflows, advances in data management and data standards must also contribute.\nDescribing and sharing analysis workflows is a great step forward for the bioimage analysis community, and this paper shows that this field is maturing and gaining good traction.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] }, { "id": "84664", "date": "04 Jun 2021", "name": "Assaf Zaritsky", "expertise": [ "Reviewer Expertise Computational cell dynamics", "data science in cell imaging", "bioimage analysis" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this Opinion Article Paul-Gilloteaux and representatives of the Network of European BioImage AnalystS (NEUBIAS) present their views on the current challenges and solution in using bioimage analysis workflows – modular image analysis pipelines that are used to process bioimaging data. Specifically, the authors survey current approaches for finding the most appropriate workflows for a given task, evaluating and comparing workflows, reproducing results, sharing code and executing workflows remotely. The opinion is dealing with a timely and important topic, and is mostly well written and easy to follow and an enjoyable read.\nThe authors present several alternative solutions for each challenge, highlighting the contribution made by NEUBIAS. This is totally legitimate since this is an opinion piece written from NEUBIAS perspective. However, I do think this point should be emphasized by including the Network of European BioImage AnalystS (NEUBIAS) in the title, by providing a brief background description about this community and by providing explicit information regarding NEUBIAS members contribution (e.g., mentioning that BIAFLOWS was developed by a NEUBIAS member).\nIn my opinion, the ideas presented in the third section (“Towards findability and accessibility of image analysis workflows”) could be integrated in the previous sections. This will improve the flow of the text without losing any content. I do not see any conceptual advantage of having a separate section as in the current form.\nPerhaps the authors would consider including some of the recent platforms that make machine/deep learning applications accessible to users such as ImJoy and ZeroCostDL4Mic? It is perhaps worth mentioning the uniqueness of machine learning-based components where training is dependent on large amounts of data, but the resulting model can be lightly disseminated (however re-training with new data is adding another layer of complexity related to parameter settings in traditional workflows).\n\nAnother related idea that NEUBIAS is heavily involved in is training (for components development, workflow reconstruction, usage and disseminations). Perhaps the authors would be interested in including some ideas in their opinion? I recently discussed related topics1.\nMore specific comments, opinions and suggestions:\nThe Abstract and the beginning of the Introduction are identical. I recommend making them separate entities, where the Abstract summarizes the main ideas, while the Introductions provides more extended background and context for the rest of this piece. In the Abstract I suggest to start with the context, move to presenting NEUBIAS and workflows (do not forget to briefly explain what is a workflow) and finish with the content of this paper (I would remove the sentence on F1000 gateway, why is it relevant in the Abstract or at all?).\nPage #3 (Introduction): Since component is the building block of a workflow, I recommend defining component before workflow.\nPage #3: “We believe that while this can be a good starting point it may not be sufficient”, can you briefly mention why this is not sufficient?\nPage #4: “the natural question is then if it is the best solution for the particular task one wants to solve”. I think that most users will not find this a “natural” question, rather, their goal is to find a “good-enough” workflow to answer the question they are interested in. Page #5 (“Toward reproducibility”): perhaps it is worth mentioning and citing the recent paper on integrating ImageJ and CellProfiler2 and/or a recent opinion reflecting on some of the aspects discussed in this section3?\nMinor formatting issue: the reference comes after the period (“.”) or comma (“,”) in several locations (e.g., refs #5-7, #10, #16, #18, #24,#26).\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-320
https://f1000research.com/articles/10-318/v1	23 Apr 21	{ "type": "Research Article", "title": "Jellyfish (Aurelia aurita) collagen scaffolds potential in alveolar bone regeneration", "authors": [ "Ranny Rachmawati", "Mohammad Hidayat", "Nur Permatasari", "Sri Widyarti", "Mohammad Hidayat", "Nur Permatasari", "Sri Widyarti" ], "abstract": "Background: Collagen scaffold is one of the most important parts of bone engineering especially for the alveolar bone regeneration. Jellyfish Aurelia aurita is one of the marine animals with the most potential for the development of collagen scaffold due to its high content of collagen and amino acid. This research aimed to evaluate the potential of jellyfish A. aurita collagen scaffold as a supporter for alveolar bone regeneration. Methods: Collagen was extracted from jellyfish by sonication method while scaffold was prepared by freeze-drying method, and chemical cross-linking with N-(3-dimethyl aminopropyl)-N’-ethyl-carbodiimide hydrochloride (EDC). Scaffold was characterized using Scanning Electron Microscope (SEM), Energy Dispersive X-Ray (EDX) spectroscopy, Fourier Transformed Infra-Red (FTIR), Cytotoxicity test with human mesenchymal stem cells (hMSCs), and biodegradability test. Results: The scaffold has a porous surface with a slightly rough texture and it has pores with size 159.9mm – 325.6mm. Carbon and oxygen are the highest elements in the scaffold. Based on FTIR results, the collagen scaffold with EDC has a lower infrared absorption rate compared to collagen scaffold without EDC. The result in cytotoxicity test showed that collagen scaffold that had been combined with EDC was not toxic to human mesenchymal stem cells (hMSCs) and even able to increase the cells growth. The scaffold with EDC has been degraded slower compared to the scaffold without EDC. Conclusion: The collagen scaffold is appropriate with the requirements as the biomaterial in supporting the alveolar bone regeneration, by fulfilling the following criteria, such as biocompatible, biodegradable, ideal porous interconnection, non-toxic, and cell viability support.", "keywords": [ "collagen scaffold", "jellyfish Aurelia aurita", "alveolar bone", "regeneration", "hMSCs" ], "content": "Introduction\n\nThe alveolar bone is a part of the maxilla and mandible bones that support the formation of dental sockets (alveoli)1. The alveolar bone is formed during an eruption of a tooth that serves to provide a mounting place for the formation of periodontal ligaments. Etiology of alveolar bone damage can occur in two categories, namely infectious and non-infectious. Damage to the alveolar bone that occurs due to infection is usually caused by anaerobic gram-negative bacteria such as Porphyromonas gingivalis, Prevotella intermedia, Agregatibacter actinomycetemcomitans. Infectious diseases of dental support tissue are commonly called periodontitis. In addition to periodontitis, tooth bone damage can be caused by mechanical trauma in the form of occlusion trauma, post-tooth extraction, and post-use orthodontic wire due to excessive pressure. The result of alveolar bone damage is the onset of a defect in the alveolar bone that results in teeth faltering and eventually loss2,3.\n\nRegeneration therapy is needed to prevent tooth loss by regrowing alveolar bone. Regenerative periodontal tissue healing occurs through the formation of new periodontal tissues, i.e. the formation of alveolar bones, the functional periodontal ligaments, and the new cementum. The indicators of bone regeneration process are increased of the differentiation and the number of bone cells (osteoblast cells)4.\n\nOne of the critical stages in the development of artificial bone tissue is the design and the manufacture of a scaffold. A scaffold is a porous biodegradable material that has a 3-dimensional structure. Scaffold serves as a structural supporter of cells, and it becomes an extracellular matrix during the natural process of bone regeneration and development, which will be naturally degraded later as new tissues grow. Scaffold material can be obtained from synthetic and natural sources5. Protein as collagen or fibrin and polysaccharides (such as chitosan and glycosaminoglycan (GAG) are proven to match the biocompatibility properties of cells but are problematic to the immunogenicity6,7.\n\nCollagen is the most commonly used polymer in the development of a scaffold. Collagen is a very low soluble protein that has typical triple helix characteristics and consists of two cross-linked a1(I) and a2(II) chains with hydrogen bonds between protein hydroxy and other residues. The triple helix structure consists of three dominant amino acids in which each parts contains amino acid ((-Gly-X-Y)n)8. Since Collagen naturally is able to activate immunogenic response when penetrated into the scaffold, it is then more often to be used in scaffold development9. The type of collagen that is often used is type I10,11, because its biocompatibility, chemical and physical properties are suitable for scaffold development. Type I collagen is classified as the main structural protein of the original extracellular matrix (ECM), which is beneficial for cell growth10. The scaffold can be applied as bone healing12,13 because of its ability to induce osteogenesis, osteoinduction, and osteoconduction phenomena, so that mesenchymal stem cells require scaffold as the attachment media, to proliferate, and to differentiate into osteoblast cells.\n\nDuring this time, the source of collagen used for the manufacture of scaffold comes from various organisms. The source of collagen that is usually used comes from the skin and tendons of bovine and porcine. However, collagen derived from bovine poses a risk of bovine spongiform encephalopathy infection while collagen derived from porcine sometime has become prohibited due to religious reasons14–17.\n\nSources of collagen from marine animals are more preferred nowadays because they are safer than land animals. One of the aquatic biota that has the potential to be a scaffold in Indonesia is jellyfish18. Based on the statistical data of the jellyfish production value in Indonesia in 2008, jellyfish production reached 500,000 tons per year19 and experienced an increase in 2011 up to 674,000 tons per year20. The increase of the jellyfish production value in Indonesia from 1998 to 2002 has an average of 2.46 % per year. Of all the types of jellyfish, A.aurita is the most numerous and consumed species in Indonesia that can be found in all the seawaters of Kalimantan, Sumatra, and Java Islands. Although the presence of jellyfish in Indonesia is relatively abundant, jellyfish is one group that receives less attention, so information about jellyfish biota resources is relatively limited. In addition, jellyfish not only contain collagen but are also rich in fatty acids that are useful for the human body21. The content of amino acids in the jellyfish is high so that almost all the needs of amino acids of the human body can be fulfilled by consuming jellyfish, except for methionine and histidine which are less than the provisions provided by FAO / WHO. Collagen in jellyfish has also become an available and relevant source for use as a component of matrices in tissue engineering7,14,22.\n\nAccording to previous studies, collagen contained in jellyfish has been used for a wide range of biomedical purposes. Hoyer et al.14 have used all parts of Rhopilema esculentum to extract its collagen and produce scaffold based on the principle of biometric mineralization. Collagen extracted from jellyfish was then cross-linked with 1-ethyl-3-(3 dimethyl aminopropyl) carbodiimide hydrochloride (EDC) and performed by dehydrothermal treatment (DHT)23. Collagen is considered as the gold standard for this field due to its high biocompatibility9,24. Collagen-based biomaterials are widely used for the manufacture of scaffolds intended for bone regeneration. In addition to having elastic mechanical properties, scaffold with the base material of jellyfish collagen has interconnection characteristics between its pores that allow mesenchymal stem cells to live and multiply so that it can be a good basis for osteogenic differentiation or osteogenesis14. Rhopilema esculentum was found to contain 16 types of amino acids with the most compositional sequences being glutamic acid (Glu), lysine (Lys), glycine (Gly), aspartic acid (Asp), leucine (Leu)25,26. In contrast, A. aurita was found to contain 15 types of amino acids with the most compositional sequences being glycine (Gly), glutamic acid (Glu), phenylalanine, (Phe), threonine (Thr), tyrosine (Tyr), lysine (Lys), serine (Ser). Of the entire A. aurita tissue, it has 57 mg of protein per gram of dry weight basis. The most composition in collagen content consists of glycine and other amino acids, including proline and hydroxyproline27.\n\nIn this present study, we create and develop a new biomaterial scaffold of jellyfish A. aurita collagen that is biocompatible, biodegradable, pore interconnected, non-toxic, and can support cell viability to help regenerate alveolar bone defects.\n\n\nMethods\n\nDried A. aurita was obtained from Prigi Beach, Trenggalek Regency, East Java, Indonesia. Dried jellyfish were cut into small pieces then washed using sterile ddH2O. After that, the sample was rewashed using NaOH. Then it was dissolved in the sterile ddH2O, then blended using a laboratory blender (Blender Philips) until crushed. The sample was then carried out for sonification and further centrifuged. A pellet was taken and put in a petri dish then freeze-dried to obtain the collagen in membrane form.\n\nMembrane and porous collagen scaffold was obtained after the freeze-drying process, it was then cross-linked in solution of N-(3-dimethyl aminopropyl)-N’-ethyl-carbodiimide hydrochloride (EDC). The scaffold was carefully rinsed in the sterile water then diluted in glycine solution. After that, the scaffold was rewashed with sterile water before freeze-dried again”\n\nScaffold membranes were affixed with carbon tape and coated with Aurum-Palladium (Au-Pd) prior to SEM and EDX Spectroscopy. The size of the samples used in SEM was 1 cm × 1 cm. Each sample was observed using SEM (FEI inspect S50) with 500x and 2000x magnifications to characterize the topography (surface, texture) and the morphology (pore size and pore interconnectivity). Meanwhile for EDX Spectroscopy, it was performed to measure the element combinations contained in the scaffold.\n\nThe scaffold membrane sample was crushed with mortar until it became a powder (± 2 mg) then further mixed with Kalium-bromide (KBr) until it formed pellets. Sample was observed using FTIR spectroscopy (Shimadzu IR-prestige 21).\n\nA scaffold membrane weighing 15 mg (W0) was then soaked into 4 mL of Phosphate Buffer Saline (PBS) solution in a Petri dish and incubated in a water bath at 37°C. PBS was replaced every two days. The scaffold was rinsed from PBS after nine days and then washed with water, lyophilized, and weighted (Wt). This assay was done in triplicate.\n\nPercentage water uptake = [(Wt – W0) / W0] × 100\n\nW0 and Wt – initial weight and weight after incubation for time ‘t’ respectively\n\nHuman mesenchymal stem cells (hMSCs), isolated from adipose cells, were kindly provided by Biomedic Laboratory, Brawijaya University. The treatment of hMSCs for in vitro experiments was approved by The Ethics Committee of Brawijaya University (1052-KEP-UB). The cells were maintained in 25 cm2 culture flask in complete medium, Dulbecco's Modified Eagle's Medium (DMEM) high glucose (Gibco) containing 10% fetal calf serum and 1.25% penicillin, streptomycin, ampicillin at 37°C in a humidified 5% CO2 incubator. The medium was replaced every 2–3 days and the culture process was completed at passage 5. The scaffold membrane was dissolved in a 1xPBS buffer (pH 7.4) with concentration of 5mg/ml and incubated in a shaker incubator (BJPX-103B, Biobase-USA) at 37°C for ± 60 days. When the scaffold membrane has dissolved completely, the mixture then centrifuged at 5000 rpm for 20 seconds to remove the undissolved material. The supernatant was then filtered using 0.45 μm millipore and diluted with complete medium to generate a treatment concentrations of 10; 100; and 1000 μg/ml. The cells were seeded in 96 well plate with density of 1.0 × 104 cells per well total volume of 100 μL. The cells were incubated at 37°C and 5% CO2 for 24 hours. The medium was replaced with treatment medium and incubated for another 24h at 37°C and 5% CO2. Then, the treatment medium was replaced with medium containing 10% WST-1 reagent and incubated at 37°C and 5% CO2 for 180 minutes. Absorbance was measured by a microplate reader (ELx808, BioTek-USA) at 450 nm wavelength. Percent viability was calculated based on the percentage of absorbance of treated cells to untreated cells (control).\n\nThe data normality were determined and the effect of three concentration of treatments were statistically evaluated with One-Way Analysis of Variance (ANOVA) and Tamhane using SPSS v22 with statistical significance set at p ≤ 0.05.\n\n\nResults\n\nThe use of scaffold as a brace of alveolar bones was inseparable from the topography and morphology, which includes pore size and interconnectivity of the scaffold. The topography of this scaffold shows a porous surface with a slightly rough texture. SEM observations on the scaffold membrane of A. aurita collagen obtained pore size ranged between 159.9 μm – 325.6 μm (Figure 1). The presence of porosity in the scaffold gives room for new cells to attach, proliferate, and differentiate into the new alveolar bone. This topology of jellyfish collagen scaffold is potential as a support on the development process of new cells in the alveolar bone.\n\n(A) Collagen Scaffold Membrane; The Porosity of scaffold by SEM Analysis with (B) 2000x magnification; (C) and (D) 500x magnification.\n\nThe microstructure elements contained in collagen scaffold were measured using EDX spectroscopy. As a result, microstructure elements found in the scaffold membrane are carbon (C), nitrogen (N), oxygen (O), sodium (Na), magnesium (Mg), aluminium (Al), silicon (Si), phosphor (P), sulfur (S) and calcium (Ca). The highest element found in the scaffold membrane was carbon (C) and the second highest was oxygen (O), 49.94 % and 27.16% of weight, respectively (Figure 2).\n\nAmide structure has been found in jellyfish A. aurita scaffold based on FTIR analysis (Figure 3.). In scaffold treated with EDC, there is Amide A found in 3426.10 cm-1, Amide B in 2924.66 cm-1, while Amide I, Amide II, and Amide III were found in 1655.57 cm-1, 1547.57 cm-1 and 1240.91 cm-1, respectively. In addition, in collagen scaffold without EDC, Amide A found in 3451.18 cm-1, Amide B in 2961.29 cm-1, while Amide I, Amide II and Amide III were found in 1651.72 cm-1, 1553,35 cm-1 and 1238.98 cm-1, respectively. Amide A, Amide B, and Amide II of the treated scaffold are lower than the untreated scaffold. On the other hand, Amide I and Amide III in the treated scaffold are stronger than untreated scaffold (Table 1).\n\nBiodegradablity is one of the properties that the scaffold must possess. Biodegradability in the scaffold is very important because the scaffold has a function as a temporary supporter, in which it will be naturally degraded by the time the alveolar bone has been regenerated. Scaffold natural degradation is expected to be in line with the growth of new bone cells so that it will not be degraded too fast or too late. The result of this study has showed that collagen scaffold treated with EDC was 53% degraded after the 9-days incubation period, while in the same incubation time, the untreated scaffold was 72% degraded after 9-days (Figure 4).\n\nBiodegradability of scaffold membrane (A) untreated scaffold; (B) Scaffold treated with EDC.\n\nCell cytoxicity test aims to determine whether collagen can support the cells growth. Human mesenchymal stem cells (hMSCs) were treated with collagen scaffold concentrations of 10; 100; and 1000 μg/ml, respectively (Figure 5). The viability cells were shown as a percentage of treated cells toward untreated control cells. The untreated control group with 0 μg/ml collagen-EDC showed 100% viable cells, while the treated groups with 10 μg/ml, 100 μg/ml and 1000 μg/ml collagen-edc treatment showed 100.03% ±1.32%, 102,686 ± %, and 109,623 ±3.42% viable cells respectively.\n\nOn the bar chart, × shows the dosage of each treatment while Y shows the %viable cells of each dose group (Figure 6). Statistical results using one-way ANOVA and Tamhane showed significant differences in concentrations between all groups (0 μg/ml, 10 μg/ml and 100μg/ml) to 1000 μg/ml (p ≤ 0.05), while each concentrations of 0 μg/ml to 10 μg/ml, 0 μg/ml to 100 μg/ml and 10 μg/ml to 100 μg/ml showed no significant difference (p>0.05) (Figure 6).\n\n\nDiscussion\n\nIn the medical sector, the scaffold has an important function especially for the treatment of new cell formation. Scaffold creation must have specific characteristics to support the quality during its application. Biocompatible, bioresorbable, and non-immunogenic properties become important requirements for scaffold manufacturing30. A scaffold of collagen jellyfish is expected to be applied as one of the options for eco-friendly and good quality treatment. Advantages of using collagen as a scaffold material are due to its biocompatible and bioactivity properties. The function of collagen as new cell growth container and to be able to facilitate cell differentiation is expected to enhance cell regeneration in alveolar bones.\n\nThe topography of the scaffold from jellyfish collagen with EDC as a cross-linker in this study shows a porous and a slightly rough surface. This topography is different from the type 1 collagen structure that usually has fibrillar structure. Adding EDC gives the binding effect of collagen structure and reconstruct it to porous form. Regarding the cell to material interaction, it is important to increase cell attachment and this depends on the surface topography of material. The topography of scaffold surface has a significant role in supporting cell activities, such as initiation of cells attachment, the proliferation of cells, and differentiation of cells. The morphology of jellyfish collagen scaffold has as a characteristic a porosity rate of ± 159.9 – 325.6 μm. According to Zhang et al.31, collagen scaffold, which has good interconnectivity, generally has a porosity rate of 45 – 800 μm. Interconnectivity of a good collagen scaffold for tissue engineering usually has a collagen scaffold with a porosity of 100–200 μm32. Proportional porosity and adequate pore size are necessary to facilitate nutrient diffusion of the entire structure of the cell so that cells can be proliferated and differentiated correctly33.\n\nElements of collagen scaffold found in this research have a higher carbon compound. Based on the research, carbon can be used to increase the value of scaffold material. Carbon in some treatment will be good in strength and stability. As scaffold material, carbon has been classified as a non-degradable scaffold. So, it can not be degraded over bone growth, but it can be a real bone substitute34. Oxygen is also having an important role in cell survival. Oxygen is needed to avoid cell hypoxia condition during the cell migration in the scaffold. Oxygen will be diffused in the cells during the respiration activity and it will also help the cells to produce ATP. The lower oxygen in tissue, namely hypoxia condition, can damage the tissue. The lower oxygen in environments will cause the production of reactive oxygen species (ROS) which can further damage the DNA by causing mutation so that it will be harmful to regenerate new cells35. In bone generation, calcium (Ca) and phosphor (P) have a close interaction. P and Ca will bind with each other and form a CaP that has a function to help the proliferation and differentiation of cells. Interaction between Ca and P will induce osteoclast and osteoblast in bone generations. While calcium phosphate is widely used in bone regeneration material like scaffold, cement, and coating36.\n\nBased on FTIR results, the collagen scaffold plus EDC has a lower infrared absorption rate compared to untreated collagen scaffolds. In this research, Amide A, Amide B, and Amide II of treated scaffold absorb lower infrared waves compared to the untreated scaffold. The differences in absorption of group function is due to the addition of EDC, indicating an interaction between the functional group in collagen (scaffold) and the functional group in EDC. EDC is an agent of imide-based zero-length cross-linker that has a functional group RN=C=NR which is known to be effective in inhibiting the enzyme degradation process of denude collagen fibrils and increasing bonds in the collagen. Besides, the functional group of EDC is known to be able to act with carboxyl ionized clusters in proteins forming O-acylisourea that can bind to protein components so that covalent amino acid chain bonds form in two more stable proteins and produce products in the form of urea. The addition of EDC to stop exposed matrix metalloproteinases (MMPs) bound to the collagen matrix is likely to occur due to the changes from the catalytic domain to allosteric23.\n\nBesides giving an effect on IR absorption in FTIR analysis, EDC also gives an effect in biodegradability of the scaffold. Biodegradability is also an important factor as the scaffold is supposed to be absorbed by surrounding tissues, without repeating the surgery process. The time needed by the scaffold to be degraded must be the same with tissue development period. So that, when the cells develop the collagen matrix, the scaffold can be provided as a structural support and then degraded after the tissue has been developed32. The addition of EDC to the scaffold is known to increase the stabilization of the collagen matrix and to lengthen the degradation period37,38. This is proven in this study where scaffold with EDC showed lower degradation compared to the untreated scaffold i.e. 53% to 72% after the 9th day of incubation, respectively (Figure 4).\n\nWST-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulphonelyl)-2H tetrazolium monosodium salt) can be converted into a water-soluble formazan by mitochondrial dehydrogenase enzymes of alive cells. The water-soluble salt is then released into the culture medium producing a color change which shows the amount of mitochondrial dehydrogenase in cell culture. Thus, the assay represents the viable cells through their metabolic activity. The result showed that collagen scaffold that had been combined with EDC was not toxic to hMSCs and even able to increase the cells growth (Figure 5 and Figure 6). This result is in accordance with reports of EDC which have showed it to be not toxic in fibroblastic L929 cell38 and increasing cells growth without being toxic39. The statistical analysis showed to be not toxic on 10 μg/ml concentration but the cell growth was insignificant compared with the ones on 100 μg/ml and 1000 μg/ml concentrations. On 1000 μg/ml concentration, the cell growth has the highest result.\n\n\nConclusion\n\nAs a conclusion, we assumed that collagen scaffold of jellyfish A. aurita which use EDC as a cross-linker has a good quality for scaffold manufacturing. The collagen scaffold from jellyfish A. aurita is appropriate with the requirements as the biomaterial in supporting the alveolar bone regeneration process, by fulfilling the following criteria, such as biocompatible, biodegradable, having ideal porous interconnection, being non-toxic, and able to support cell viability. In order to get more optimal scaffold development results, further research focusing on in vivo models will be useful in the future.\n\n\nData availability\n\nZenodo: Jellyfish (Aurelia aurita) collagen scaffolds potential in alveolar bone regeneration, https://doi.org/10.5281/zenodo.445004040.\n\nThis project contains the following underlying data:\n\n- Uncropped/unedited electron microscopy images showing morphology of jellyfish A.aurita collagen scaffold (500x magnification)\n\n- Uncropped/unedited electron microscopy images showing morphology of jellyfish A.aurita collagen scaffold (2000x magnification)\n\n- Raw data of Energy Dispersive X-ray (EDX) spectroscopy showing elements contained in a jellyfish A.aurita collagen scaffold\n\n- Unedited Fourier Transformed Infrared (FTIR) images showing wavelength of jellyfish A.aurita collagen scaffold without EDC (untreated scaffold)\n\n- Unedited Fourier Transformed Infrared (FTIR) images showing wavelength of jellyfish A.aurita collagen scaffold with EDC (treated scaffold)\n\n- Raw data of biodegradable test result\n\n- Raw data of absorbance reading for WST assay for hMSCs\n\nZenodo: Jellyfish (Aurelia aurita) collagen scaffolds potential in alveolar bone regeneration, http://doi.org/10.5281/zenodo.443116041.\n\nThis project contains the following extended data:\n\n- Uncropped/unedited electron microscopy images showing morphology of jellyfish A.aurita collagen scaffold (500x magnification)\n\n- Uncropped/unedited electron microscopy images showing morphology of jellyfish A.aurita collagen scaffold (2000x magnification)\n\n- Uncropped/unedited electron microscopy images showing morphology of jellyfish A.aurita collagen scaffold (20.000x magnification)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgments\n\nSpecial thanks to Rizka Pantris and Marlita from Biomolecular Laboratory for their enlightening, encouraging discussions, and support. We also thank Sinta Husada and Saiful from Biomedic Laboratory for providing hMSCs. The last, thank Yuanita Lely for the discussion and support the statistical analysis.\n\n\nReferences\n\nSheikh Z, Sima C, Glogauer M: Bone Replacement Materials and Techniques Used for Achieving Vertical Alveolar Bone Augmentation. Materials (Basel). 2015; 8(6): 2953–2993. Publisher Full Text \| Free Full Text\n\nCarranza FA, Camargo PM, Takei HH: bone loss and patterns of bone destruction. In: Carranza’s Clinical Periodontology. 2012; 140–150. Publisher Full Text\n\nMichael GN, Takei H, Klokkevold P, et al.: Newman and Carranza’s Clinical Periodontology. (Elsevier). 2018. Reference Source\n\nBaghban AA, Dehghani A, Ghanavati F, et al.: Comparing alveolar bone regeneration using Bio-Oss and autogenous bone grafts in humans: a systematic review and meta-analysis. Iran Endod J. 2009; 4(4): 125–130. PubMed Abstract \| Free Full Text\n\nMa PX: Scaffolds for tissue fabrication. Mater Today. 2004; 7(5): 30–40. Publisher Full Text\n\nPuppi D, Chiellini F, Piras AM, et al.: Polymeric materials for bone and cartilage repair. Prog Polym Sci (Oxford). 2010; 35(4): 403–440. Publisher Full Text\n\nMa PX, Elisseeff J: Scaffolding in tissue engineering. (Boca Raton: CRC Press). 2005. Publisher Full Text\n\nSchmidt MM, Dornelles RCB, Mello RO, et al.: Collagen extraction process. Int Food Res J. 2016; 23(3): 913–922. Reference Source\n\nFelician Z, Xia C, Qi W, et al.: Collagen from Marine Biological Sources and Medical Applications. Chem Biodivers. 2018; 15(5): e1700557. PubMed Abstract \| Publisher Full Text\n\nAllam E, et al.: Collagen scaffolds: Tissue engineering and repair. In: Type I Collagen: Biological Functions, Synthesis and Medicinal Applications. 2012.\n\nRastian Z, Pütz S, Wang YJ, et al.: Type I collagen from jellyfish Catostylus mosaicus for biomaterial applications. ACS Biomater Sci Eng. 2018; 4(6): 2115–2125. PubMed Abstract \| Publisher Full Text\n\nVelasco MA, Narváez-Tovar CA, Garzón-Alvarado DA: Design, materials, and mechanobiology of biodegradable scaffolds for bone tissue engineering. Biomed Res Int 2015; 2015: 729076. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang D, Wu X, Chen J, et al.: The development of collagen based composite scaffolds for bone regeneration. Bioact Mater. 2017; 3(1): 129–138. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHoyer B, Bernhardt A, Lode A, et al.: Jellyfish collagen scaffolds for cartilage tissue engineering. Acta Biomater. 2014; 10(2): 883–892. PubMed Abstract \| Publisher Full Text\n\nGomez-Guillen MC, Giménez B, López-Caballero LE, et al.: Functional and bioactive properties of collagen and gelatin from alternative sources: A review. Food Hydrocoll. 2011; 25(8): 1813–1827. Publisher Full Text\n\nAzizur Rahman M: Collagen of extracellular matrix from marine invertebrates and its medical applications. Mar Drugs. 2019; 17(12): 118. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSilvipriya KS, Kuma KK, Bhat AR, et al.: Collagen: Animal sources and biomedical application. J Appl Pharm Sci. 2015; 5(3): 123–127. Publisher Full Text\n\nYusuf S, Fahmid IM, Abdullah N, et al.: Indonesian jellyfish as potential for raw materials of food and drug. In: IOP Conf Ser: Earth Environ Sci. 2018; 157(1): 012056. Publisher Full Text\n\nPerikanan KK: Laporan Akuntabilitas Kinerja Instansi Pemerintah (LAKIP). 2008.\n\nPerikanan KK: Laporan Akuntabilitas Kinerja Instansi Pemerintah (LAKIP). 2011. Reference Source\n\nRichardson AJ, Bakun A, Hays GC, et al.: The jellyfish joyride: causes, consequences and management responses to a more gelatinous future. Trends Ecol Evol. 2009; 24(6): 312–22. PubMed Abstract \| Publisher Full Text\n\nGershwin LA: Systematics and biogeography of the jellyfish Aurelia labiata (Cnidaria: Scyphozoa). Biol Bull. 2001; 201(1): 104–119. PubMed Abstract \| Publisher Full Text\n\nTurco G, Frassetto A, Fontanive L, et al.: Occlusal loading and cross-linking effects on dentin collagen degradation in physiological conditions. Dent Mater. 2016; 32(2): 192–199. PubMed Abstract \| Publisher Full Text\n\nSong E, Kim SY, Chun T, et al.: Collagen scaffolds derived from a marine source and their biocompatibility. Biomaterials. 2006; 27(15): 2951–2961. PubMed Abstract \| Publisher Full Text\n\nKhong NMH, Yusoff FM, Jamilah B, et al.: Nutritional composition and total collagen content of three commercially important edible jellyfish. Food Chem. 2016; 196: 953–960. PubMed Abstract \| Publisher Full Text\n\nYu H, Yusoff FM, Li R, et al.: Amino acid composition and nutritional quality of gonad from jellyfish Rhopilema esculentum. Biomedicine Preventive Nutrition. 2014; 4(3): 399–402. Publisher Full Text\n\nLeone A, Lecci RM, Durante M, et al.: The bright side of gelatinous blooms: Nutraceutical value and antioxidant properties of three Mediterranean jellyfish (Scyphozoa). Mar Drugs. 2015; 13(8): 4654–4681. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBarzideh Z, Latiff A, Gan CY, et al.: Isolation and characterisation of collagen from the ribbon jellyfish (Chrysaora sp.). Int J Food Sci Technol. 2014; 49(6): 1490–1499. Publisher Full Text\n\nWang Y, Manh NV, Wang H, et al.: Synergistic intrafibrillar/extrafibrillar mineralization of collagen scaffolds based on a biomimetic strategy to promote the regeneration of bone defects. Int J Nanomedicine. 2016; 11: 2053–2067. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHorst M, Madduri S, Gobet R, et al.: Scaffold Characteristics for functional hollow organ regeneration. Materials. 2010; 3(1): 241–263. Publisher Full Text \| Free Full Text\n\nZhang L, Morsi YY, Wang Y, et al.: Review scaffold design and stem cells for tooth regeneration. Jpn Dent Sci Rev. 2013; 49(1): 14–26. Publisher Full Text\n\nSatish Kumar T, Vijaya Ramu D, Sampath Kumar NS: Preparation and characterization of biodegradable collagen-Chitosan scaffolds. Mater Today Proc. 2019; 19: 2587–2590. Publisher Full Text\n\nLanza R, Langer R: Principles of Tissue Engineering. (Amsterdam: Elsevier Inc). 2010.\n\nTanaka M, Aoki K, Haniu H, et al.: Applications of carbon nanotubes in bone regenerative medicine. Nanomaterials (Basel). 2020; 10(4): 659. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGholipourmalekabadi M, Zhao S, Harrison BS, et al.: Oxygen-Generating Biomaterials: A New, Viable Paradigm for Tissue Engineering? Trends Biotechnol. 2016; 34(12): 1010–1021. PubMed Abstract \| Publisher Full Text\n\nJeong J, Kim JH, Shim JH, et al.: Bioactive calcium phosphate materials and applications in bone regeneration. Biomater Res. 2019; 23: 4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPark SN, Park JC, Kim HO, et al.: Characterization of porous collagen/hyaluronic acid scaffold modified by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide cross-linking. Biomaterials. 2002; 23(4): 1205–1212. PubMed Abstract \| Publisher Full Text\n\nScheffel DLS, Hebling J, Scheffel RH, et al.: Stabilization of dentin matrix after cross-linking treatments, in vitro. Dent Mater. 2014; 30(2): 227–233. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLehmann N, Christ T, Daugs A, et al.: EDC Cross-linking of Decellularized Tissue: A Promising Approach? Tissue Eng Part A. 2017; 23(13–14): 675–682. PubMed Abstract \| Publisher Full Text\n\nRachmawati R, Hidayat M, Permatasari N, et al.: Jellyfish (Aurelia aurita) collagen scaffolds potential in alveolar bone regeneration [Data set]. Zenodo. 2021. http://www.doi.org/10.5281/zenodo.4431156\n\nRachmawati R, Hidayat M, Permatasari N, et al.: Jellyfish (Aurelia aurita) collagen scaffolds potential in alveolar bone regeneration [Data set]. Zenodo. 2021. http://www.doi.org/10.5281/zenodo.4431160" }	[ { "id": "86750", "date": "06 Jul 2021", "name": "Ika Dewi Ana", "expertise": [ "Reviewer Expertise Bioceramics", "Tissue engineering", "Regenerative dentistry", "Lab-on-a-chip" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a good research report. My concerns are:\n1. How did author identify the A. aurita? Is there any biological approach to identify it to obtain homogenous source of A. aurita? Please explain in the text.\n2. Why did author choose EDC among other crosslinking agents? Please explain in the text.\n3. Cell adhesion test cannot exactly predict in vivo results. If the author can add data on protein adsorption, that would be better. For this purpose, please refer to:\n\nJanuariyasa et al. (20201) Sari et al. 2021 (20212) Sari et al. 2021 (20213)\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "92622", "date": "21 Jun 2024", "name": "Mike Barbeck", "expertise": [ "Reviewer Expertise Bone tissue engineering", "biomaterial development" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMany thanks for the interesting article. Although I like the manuscript in general, different things should be revised prior to indexing.\nWhat about the bio-incompatibility of EDC as a crosslinking agent? Please discuss. Why did you use even this crosslinker?\n\nPlease discuss your results even in view of the following article: \"In Vivo Analysis of the Biocompatibility and Immune Response of Jellyfish Collagen Scaffolds and its Suitability for Bone Regeneration\" (Flaig et al., 20201). In this context, please discuss your results of biodegradability etc.\n\nWhy did you use hMSCs for biocompatibility analysis? What about the differentiation of these cells onto jellyfish scaffolds?\n\nPlease include a normal graph instead of the PowerPoint slide in Fig. 4. What about the number of specimens analyzed and the standard deviation?\n\nWhat about significant differences for the cell viability for the cell viability measurements? Why did you use ANOVA + Tamhane test? Please explain.\n\nPlease discuss your results in context of important factors of tissue engineering such as vascularization and pore size but also for osteoblasts or osteoconduction.\n\nIn general, your results does not allow to give any answer on the suitability of these scaffolds for alveolar bone regeneration. Which result let you think they are suitable for such an application? In this context, it is necessary to use osteoblasts for the in vitro analysis or to measure the expression of relevant protein expression by hMSCs, please explain.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/10-318
https://f1000research.com/articles/9-327/v2	27 Aug 20	{ "type": "Research Article", "title": "Revisiting early-stage COVID-19 strategy options", "authors": [ "Philip Machanick" ], "abstract": "Background: Early-stage interventions in a potential pandemic are important to understand as they can make the difference between runaway exponential growth that is hard to turn back and stopping the spread before it gets that far. COVID19 is an interesting case study because there have been very different outcomes in different localities. These variations are best studied after the fact if precision is the goal; while a pandemic is still unfolding less precise analysis is of value in attempting to guide localities to learn lessons of those that preceded them. Methods: I examine three factors that could differentiate strategy: asymptomatic spread, differences in use of the Bacillus Calmette-Guerin (BCG) tuberculosis vaccine and cloth face masks. Results: Differences in disease progression as well as the possibility of alternative strategies to prevent COVID-19 from entering the runaway phase or damping it down later can be elucidated by a study of asymptomatic infection. A study to demonstrate not only what fraction are asymptomatic but how contagious they are will also inform policy on universal mask wearing. Conclusions: While a COVID-19 outbreak is at a level that makes accurate trace-and test possible, investigation of asymptomatic transmission is viable and should be attempted to enhance understanding of spread and variability in the disease as well as policy options for slowing the spread.", "keywords": [ "COVID-19", "early-stage pandemic intervention", "asymptomatic transmission" ], "content": "Introduction\n\nAs the COVID-19 pandemic has spread, its outcomes have differed by locality. In some, it has been contained quickly. In others, the rapid growth has slowed but not stopped. In many, the rapid growth has driven health systems to the point of collapse. New York state is the epicenter of the United States epidemic. What adds urgency to the search for alternative containment strategies is the fact that mortality rate (deaths as a fraction of population) in New York state on 22 April 2020 surpassed 1000 per million. If scaled to the entire country, this would be over 300,000 deaths1. At time of writing the second version of this paper, US deaths are at almost 180,000, illustrating that there is still work to be done at containment.\n\nSince the pandemic is still playing out it is useful to reflect on the positive and negative outcomes and to try to map a way ahead for localities where it has not gone past the stage where it can easily be contained. Given that no known remedy exists for the disease, and that it is spreading too fast to rely on a vaccine to avoid major health or economic problems, non-pharmaceutical interventions (NPI) are the most critical thing to get right. Now that the pandemic has progressed beyond its initial stages, understanding asymptomatic transmission remains important as the question of universal mask wearing remains controversial1 with inadequate testing of informal masks nonetheless at least justifying their use in terms of the precautionary principle2. One study shows that viral shedding starts 2 days or more before symptoms show and that infectiousness peaks 1–2 days prior to symptoms and presymptomatic transmission is 37% to 48% and that this figure can be as high as 62% without adequate case finding. There are no definitive studies of asymptomatic transmission, despite evidence that it is real3.\n\nWhile understanding asymptomatic transmission alone does not fill the gap in investigating the efficacy of informal masks, it strengthens the case for applying the precautionary principle pending such a study. Studying informal masks is inherently difficult as there is so much variability, which is why I propose an initial focus on asymptomatic infection. Asymptomatic infection can also assist with understanding the highly variable progression of the disease.\n\nA number of NPIs have been tried from social distancing to complete lockdowns. The consequence of acting too slowly is the risk of crashing a health system, which has hit some of the best in the world (Italy for example has historically been near the top of the World Health Organization’s ranking4; and the epicentre of the COVID-19 outbreak is in the north5, which has Italy’s strongest health resources6. Though the pandemic at time of writing is still developing, it is worth reviewing options for countries able to avoid runaway exponential growth.\n\nBy way of example, I looked at options for South Africa, which embarked on a 21-day lock down7 that started at about the time when 1,000 cases were reported (midnight, 26 March 2020; subsequently extended by another two weeks to 30 April), in the first version of this paper. At this much later stage of progress of the pandemic, countries where cases are declining have the same opportunity to use spare testing capacity for a study I propose in this paper.\n\nI examine case studies in other localities encompassing the variability in outcomes and assess likely contributing factors to this variance. Given the shortness of time to make decisions, I do not attempt to develop a rigorous model but rely on extracting meaning from these case studies.\n\nIn my opinion, ignoring asymptomatic spread is a major error; a relatively simple experiment in a country like South Africa at the early stage of spread (or now at a later stage as outlined above) could validate this opinion. If proved correct, many lives could be saved. If proved incorrect, the cost is low relative to the benefit. I therefore urge that the experiment be carried out as a matter of urgency.\n\nIt is natural at an early stage of a rapidly-expanding pandemic to focus on the most serious cases as these are the ones where interventions make the biggest difference. Now that there is more time to assess evidence, there is also a case to do detailed studies of less serious cases to understand better what predicts progression to the worst effects. Identifying asymptomatic cases in particular could aid with this as they represent the extreme of the mild form of the disease.\n\nIn the remainder of the paper, I describe significant unknowns, work through cases studies and examine other factors, leading to the conclusion that a study of asymptomatic spread is the most urgent gap in current knowledge that must be addressed to assess options for containment. I propose a strategy for identifying and investigating asymptomatic cases.\n\n\nSignificant unknowns\n\nBecause testing started under pressure, standards are not consistent8. That means statistics like case fatality rate are problematic to compare across localities.\n\nAnother big unknown is the true number of cases since many that were not serious enough to require hospitalization may have resolved without being counted where testing was inadequate; if the asymptomatic fraction is as high as claimed in some instances, that also skews the case fatality rate high.\n\nAll of these factors also result in difficulty in establishing an accurate value for the basic reproduction rate R0, the initial mean number of infections per infected case. The value of R0 matters for computing the herd immunity level, widely reported in the mass media as 60% of the population, the number used to justify the initial British response of allowing it to run through the population9.\n\nHerd immunity occurs when the fraction of the population immunised (either by vaccination or by acquiring immunity post-infection) exceeds the threshold Pherd in Equation 1,\n\nFigure 1 illustrates how Pherd varies with R0 (assuming E = 1; in the absence of a vaccine, this means that any recovered cases cannot be reinfected). For seasonal influenza, if R0 = 1.3, the herd immunity threshold is 23%. For R0 = 2.5, the herd immunity threshold is 60%. However, if the true COVID-19 R0 value is significantly higher, so is the the herd immunity threshold. For example, if R0 = 4, Pherd is 75%.\n\nIf R0 for COVID-19 is 2.5, herd immunity occurs after 60% of the population is infected (green arrow). The mean R0 value for seasonal influenza results in herd immunity at about 23% of the population (red arrow).\n\nEven for influenza, R0 can vary widely depending on the strain. For the H1N1 strain, R0 was estimated at 1.4–1.6; for the 1918 flu, the estimated R0 range is 1.4–2.8 and even seasonal flu has a wide R0 range of 0.9–2.111.\n\nOne study reports COVID-19 R0 values varying from 1.4 to 3.812. Another narrows the range to 2.24 to 3.5813. A model with R0 = 2.68 yields a doubling time of 6.4 days14. The doubling time was far shorter than this during peak growth in places where it was not under control. In the United States, for example, doubling time was less than 3 days 1–24 March 2020 (see Figure 2, based on the rule-of-7015). That rapidity of growth suggests that R0 is on the high rather than low side of published estimates, but the low level of testing in the USA makes it difficult to derive robust measures.\n\nFor much of the month, doubling time is 2–3 days.\n\nWith so much uncertainty, relying on herd immunity is folly, as was discovered in the UK9.\n\nBroadly speaking, countries with a tradition of personal liberty and rejecting authority have found it hard to adapt to NPIs like social distancing16. Informal settlements and other high-density dwellings of the poor also make social distancing hard17. For this reason, it is important to explore all alternatives including those that were missed in early stages so that other countries that are being hit later can learn the right lessons.\n\n\nMethods\n\nI use Python 3.7.4 within the Jupyter Notebook environment version 6.0.3 to do anyalysis. All the code used is archived on GitHub18 and is labelled as version 1.1.1.\n\nData is from the Worldometers web site and papers cited; data used in analysis is embedded in the GitHub archive18. A snapshot of the Worldometers site containing the data used is archived at Webarchive (updated in Webarchive for Version 2 of the paper).\n\nHerd immunity is calculated based on the standard formula assuming exponential growth using Equation 2.\n\nI calculate doubling time for March 2020 using the rule-of-70, which assumes exponential growth and is accurate under that assumption:\n\n\nCase studies\n\nI consider three cases: instances where the disease has run its course, examples where containment has slowed the growth to the point of being manageable and finally examples where growth has severely stressed health systems.\n\nThese examples are not meant to be exhaustive but provide archetypes of different types of outcome.\n\nI use two types of localities as examples of where the disease has effectively run to completion: cruise liners and a village in Italy, Vo’Euganeo.\n\nI take Diamond Princess, a widely cited case, as representative of the first kind.\n\nDiamond Princess is one of the most documented examples. Of the 3,711 passengers and crew on board, 634 tested positive out of the 3,063 tested and 328 were asymptomatic19 – meaning that over 50% were asymptomatic.\n\nWhat is startling about these numbers is how few tested positive. Given the constrained environment of a cruise liner, a higher rate of social mixing would be expected than in normal living conditions.\n\nHowever the major figure to take way from this is the fact that over half were asymptomatic.\n\nFor the Italian village, the number of cases that tested positive but were asymptomatic were in the range of 50–75%2 20.\n\nWhat is particularly noteworthy is that 100% of the population was tested so no asymptomatic cases were missed. This completeness of coverage does not guarantee that a similar result would occur in a different population but at least provides a clear data point. All positive cases were isolated. The number who were ill fell from 88 to 7 in 10 days.\n\nBy contrast with this strategy, on 25 February 2020, the strategy for the rest of Italy changed from broad testing of all contacts to a focus on only testing those who had clear symptoms and required hospitalization21. This decision was taken when the country had less than 100 cases.\n\nIt is a puzzle why it should be more important to test cases who must be hospitalized anyway rather than find those in the community who may be contagious.\n\nI quote the advice of Italian immunologist Prof Romagnani (via Google Translate):\n\nIt is therefore absolutely essential to extend the swabs to the majority of the population, in particular to risk categories (i.e. exposed to multiple contacts), and therefore isolate the virus positive subjects and their contacts, even if asymptomatic, as early as possible. In particular, it is absolutely necessary to swab all those who have a high probability of transmitting the virus, especially if they live in closed communities with multiple and close contacts. Finally, it is very important that all those at risk wear masks, not so much to protect themselves from infection, but rather to protect others from themselves, even when they do not show symptoms.\n\nEven if we take the lower figure of 50% of the Vo’Euganeo being asymptomatic, ignoring this factor in testing is a major error. However, in a larger population it is not practical to test everyone and not necessarily useful as a person tested negative could subsequently be infected. Instead, treating everyone exposed to a known case as infected until proved otherwise would catch a significant fraction of the asymptomatic cases.\n\nSeveral countries have managed to contain exponential growth after a major outbreak. Two examples are China and South Korea.\n\nSince China had the first outbreak, they did not act fast enough and had to close down a major part of their economy. Much of Hubei province was effectively placed under quarantine on 23 January 2020, using methods such as tracking social media to enforce it. Since China is a large country, it was possible to isolate the infected region and rush in resources from the rest of the country. Overall the measures used seem unlikely to be transferable to most other countries22.\n\nSouth Korea instead took an approach of rapid and comprehensive testing while isolating all known cases and all their contacts23. With no lockdown, they brought the increase off the exponential trend. That supports my opinion of how the Italian test strategy went wrong.\n\nThe United States only showed signs of breaking exponential growth after over 300,000 cases were reported on 4 April 2020. Some days during March 2020, growth was at 40% or more per day with a doubling time of two days and, up to 24 March, doubling time was generally three days or less.\n\nOver the period 2–20 March 2020, growth in the US ranged from 24% to 49% per day. With that level of growth, the United States had no option but to implement increasingly country-wide lockdowns. Other countries that resisted this strategy ran into exactly the same issue: exponential growth defeated their health systems.\n\nIn a relatively large country like the US, if growth is not even across the country, there is the option to rush resources to the hotspots as was done in China. However, this option is not as attractive as stopping the spread much earlier as the hotspots become extremely resource intensive. New York state for example has been widely reported as estimating a requirement of 40,000 ventilators at peak3 though the actual peak number of cases requiring ventilation did not come close to this level24.\n\nLocalities that have suffered this sort of resource intensity have also generally had higher case fatality rates. This could in part be an artefact of inadequate testing that under-counts cases. However Italy (13% case fatality) has a higher per capita rate of testing than Germany (3.4% case fatality) as of 20 April 2020: 23,985 per million population vs. 20,629 for Germany. Under-counting asymptomatic cases is not likely to be the sole issue.\n\n\nOther factors\n\nAn important factor to consider is comorbidities. South Africa has high rates of tuberculosis and HIV infection, both of which are significant risk factors for any pulmonary disease25. In one study in Italy, out of 355 patients who died, only 3 (0.8%) had no prior condition. 25.1% had one condition, 25.6% had two and 48.5% had three or more21.\n\nBalanced against this is statistical evidence that differences in national coverage of the Bacillus Calmette-Guerin (BCG) tuberculosis vaccine explain differences in case fatality rates26. This study is not peer reviewed and therefore should not be relied on too strongly. A pure statistical study without direct causal evidence points to the need to establish causality rather than signifying causality. However, there would be merit in identifying any cohorts who missed childhood BCG vaccination and provide them with this additional extra protection, provided that it is not contraindicated. BCG is already administered on a mass scale and its risks are well understood.\n\nStudy of case outcomes in a country like South Africa where BCG is commonly administered but where there is known to be variations in geographic coverage27 could help to validate causality.\n\nWeighed against relying on BCG coverage is that Iceland, which does not have mandatory BCG vaccination28, was very successful in slowing the spread by an aggressive testing programme, including quarantining everyone who had contacted a known case29.\n\nAnother possible mitigating factor is that existing HIV remedies are among those being investigated for efficacy against COVID-1930. However, it would be foolhardy to rely on this as a mitigating factor without solid evidence that it applies widely enough to matter.\n\nFinally there is indirect evidence of asymptomatic spread in the apparent efficacy of masks in slowing spread. While early WHO advice was against the asymptomatic mask wearing, this was part of an advisory that aimed to prevent a run on medical-grade masks31. More recently there is support for cloth masks being worn by the public2 and experimental evidence that surgical masks block aerosol transmission of coronaviruses and influenza viruses32. While some still doubt the evidence, the fact that mandatory mask wearing was a factor in reducing the spread of the 1918 influenza pandemic33 supports the case for encouraging mask-wearing as a protection for the COVID-19 pandemic provided that this does not deplete supplies of medical-grade masks and there is a programme of public education on use of masks and their role in the context of other measures like distancing and hygiene.\n\nGiven the high risk associated with comorbidities, it is premature to place too much reliance on any mitigating factors other than slowing transmission. NPIs are the main game until pharmaceutical options become viable on a large scale. Since the initial version of this paper, there have been no definitive studies showing a causal link between BCG and COVID-19 outcomes. A simple alternative explanation is that the countries that were caught least prepared coincidentally did not have BCG programmes and this possibility needs to be dismissed before relying on the possibility that BCG vaccination could be beneficial34.\n\n\nProposed Research\n\nSince the biggest unknown is the prevalence and contagiousness of asymptomatic infection, I propose a project to identify such cases early and identify informative features. Features of interest include:\n\nviral shedding after initial infection; this should include variability in magnitude and duration\n\nprevalence in asymptomatic cases of comorbodities and risk factors like age\n\ntesting for antibodies including those for related but more benign coronaviruses\n\ntesting for T cell variability and other features of the immune system that may influence disease progression35\n\nTo identify these cases in time to measure viral shedding from the start of infection, comprehensive contact tracing of a representative sample of the infected population is necessary. Any who test positive out of this cohort can be followed up by a further round of contact tracing; if this finds more positive cases that have been in contact with only asymptomatic cases, that would provide a measure of asymptomatic contagion. If however the programme prevents secondary infections, viral shedding will still be a useful indicator of contagiousness. In one study, rapid identification and isolation of asymptomatic cases prevented secondary infections36 but this should not be taken as indicative that asymptomatic cases are not contagious.\n\nThis is a project that could be carried out with a modest burden on testing capacity – as long as the pandemic is not expanding at full pace.\n\n\nConclusions\n\nThe most significant finding out of this review is that we do not know enough about asymptomatic transmission. This is a problem easily remedied in early-stage spread – or at a later stage when cases are reducing – and there is spare test capacity.\n\nThe proposed project would give a clearer picture of the potential for asymptomatic spread and add to the evidence for universal mask wearing.\n\nIf the experiment to measure asymptomatic spread shows that it is a significant factor, that signifies a change in testing strategy. Should any asymptomatic positives be discovered, more aggressive action should follow: they and all their contacts should be quarantined and released when they no longer test positive, taking into account time for incubation.\n\nSouth Africa, at the date of the first version of the paper, had less than 3500 cases; at time of writing the second version, the daily number of cases had declined to 25% of the peak. Going back to test all contacts was doable at the outset; with test demand 25% lower than at peak, testing all contacts of a representative sample is practical. While it is possible that the data cited in this paper on the asymptomatic fraction is wrong, the cost of finding this out is very low compared with the cost of not containing the spread. As previously noted, Iceland does not do widespread BCG vaccination28 and yet has been successful in containing the spread. The Iceland experience indicates that there is no need to rely on questionable evidence that the BCG vaccine provides protection: act early and catch all cases including the asymptomatic and the spread can be curtailed29.\n\nThe cost of taking the step I advocate is far lower than the cost of allowing asymptomatic spread to get out of control. If it leads to an effective containment strategy, it will also remove the need for an economically damaging extended lockdown. Even at a later stage of the pandemic, informing policy choices like mask wearing is useful, as is better understanding of the drivers in variabililty of the disease. And economically damaging NPIs can be better avoided if we understand the disease better – how it spreads, what makes people more vulnerable.\n\n\nData availability\n\nCOVID-19 data used was gathered from Worldometer: https://www.worldometers.info/.\n\nData is archived at: http://web.archive.org/web/20200422135436/https://www.worldometers.info/coronavirus/ and the update for the second version is archived at http://web.archive.org/web/20200822095038/https://www.worldometers.info/coronavirus/.\n\nThe Python code used to graph herd immunity versus R0 as well graphing doubling time and the US exponential growth scenarios is available from GitHub: https://github.com/philip-mach/herd-immunity.\n\nZenodo: philip-mach/herd-immunity: includes herd immunity and doubling time graphs. http://doi.org/10.5281/zenodo.376235618.\n\nData is available under the terms of the BSD 2-clause license.", "appendix": "Footnotes\n\n1The data source for cases and fatalities, unless otherwise stated, is the Worldometers web site.\n\n2The wide range is not explained – possibly defining asymptomatic is imprecise.\n\n3For example: https://abc7ny.com/ventilators-coronavirus-new-york-ny-cases-in/6086097/.\n\n\nReferences\n\nFeng S, Shen C, Xia N, et al.: Rational use of face masks in the COVID-19 pandemic. Lancet Respir Med. 2020; 8(5): 434–436. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGreenhalgh T, Schmid MB, Czypionka T, et al.: Face masks for the public during the covid-19 crisis. BMJ. 2020; 369: m1435. PubMed Abstract \| Publisher Full Text\n\nGandhi M, Yokoe DS, Havlir DV: Asymptomatic transmission, the Achilles’ heel of current strategies to control COVID-19. N Engl J Med. 2020; 382(22): 2158–2160. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTandon A, Murray CJL, Lauer JA, et al.: Measuring overall health system performance for 191 countries. Technical report, World Health Organisation, 2000. Reference Source\n\nSantacroce L, Charitos IA, Del Prete R: COVID-19 in Italy: An overview from the first case to date. Electron J Gen Med. 2020; 17(6): em235. Publisher Full Text\n\nSignorelli C, Odone A, Oradini-Alacreu A, et al.: Universal Health Coverage in Italy: lights and shades of the Italian National Health Service which celebrated its 40th anniversary. Health Policy. 2020; 124(1): 69–74. PubMed Abstract \| Publisher Full Text\n\nHamzelou J: World in lockdown. New Sci. 2020; 245(3275): 7. Publisher Full Text\n\nKenyon C: Flattening-the-curve associated with reduced COVID-19 case fatality ratesan ecological analysis of 65 countries. J Infect. 2020; 81(1): e98–e99. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHunter DJ: Covid-19 and the Stiff Upper Lip - The Pandemic Response in the United Kingdom. N Engl J Med. 2020; 382(16): e31. PubMed Abstract \| Publisher Full Text\n\nFine P, Eames K, Heymann DL: \"Herd Immunity\": a rough guide. Clin Infect Dis. 2011; 52(7): 911–916. PubMed Abstract \| Publisher Full Text\n\nCoburn BJ, Wagner BG, Blower S: Modeling influenza epidemics and pandemics: insights into the future of swine flu (H1N1). BMC Med. 2009; 7(1): 30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFang Y, Nie Y, Penny M: Transmission dynamics of the COVID-19 outbreak and effectiveness of government interventions: A data-driven analysis. J Med Virol. 2020; 92(6): 645–659. PubMed Abstract \| Publisher Full Text\n\nZhao S, Lin Q, Ran J, et al.: Preliminary estimation of the basic reproduction number of novel coronavirus (2019-nCoV) in China, from 2019 to 2020: A data-driven analysis in the early phase of the outbreak. Int J Infect Dis. 2020; 92: 214–217. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWu JT, Leung K, Leung GM: Nowcasting and forecasting the potential domestic and international spread of the 2019-nCoV outbreak originating in Wuhan, China: a modelling study. Lancet. 2020; 395(10225): 689–697. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBermingham JR: Exponential population growth and doubling times: Are they dead or merely quiescent? Popul Environ. 2003; 24(4): 313–327. Publisher Full Text\n\nSibony AL: The UK COVID-19 response: A behavioural irony? Eur J Risk Regul. 2020; 1–11. Publisher Full Text\n\nGibson L, Rush D: Novel Coronavirus in Cape Town informal settlements: Feasibility of Using Informal Dwelling Outlines to Identify High Risk Areas for COVID-19 Transmission From A Social Distancing Perspective. JMIR Public Health Surveill. 2020; 6(2): e18844. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMachanick P: philip-mach/herd-immunity: adds doubling time graph. 2020. http://www.doi.org/10.5281/zenodo.3762356\n\nMizumoto K, Kagaya K, Zarebski A, et al.: Estimating the asymptomatic proportion of coronavirus disease 2019 (COVID-19) cases on board the Diamond Princess cruise ship, Yokohama, Japan, 2020. Euro Surveill. 2020; 25(10): 2000180. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDay M: Covid-19: identifying and isolating asymptomatic people helped eliminate virus in Italian village. BMJ. 2020; 368: m1165. PubMed Abstract \| Publisher Full Text\n\nOnder G, Rezza G, Brusaferro S: Case-Fatality Rate and Characteristics of Patients Dying in Relation to COVID-19 in Italy. JAMA. 2020. PubMed Abstract \| Publisher Full Text\n\nKupferschmidt K, Cohen J: Can China's COVID-19 strategy work elsewhere? Science. 2020; 367(6482): 1061–1062. PubMed Abstract \| Publisher Full Text\n\nCohen J, Kupferschmidt K: Countries test tactics in 'war' against COVID-19. Science. 2020; 367(6484): 1287–1288. PubMed Abstract \| Publisher Full Text\n\nUppal A, Silvestri DM, Siegler M, et al.: Critical Care And Emergency Department Response at the Epicenter of the COVID-19 Pandemic. Health Aff (Millwood). 2020; 39(8): 1443–1449. PubMed Abstract \| Publisher Full Text\n\nBoffa J, Mhlaba T, Sulis G, et al.: COVID-19 and tuberculosis in South Africa: A dangerous combination. S Afr Med J. 2020; 110(5). Publisher Full Text\n\nMiller A, Reandelar MJ, Fasciglione K, et al.: Correlation between universal BCG vaccination policy and reduced morbidity and mortality for COVID-19: an epidemiological study. medRxiv. 2020. Publisher Full Text\n\nFadnes LT, Jackson D, Engebretsen IM, et al.: Vaccination coverage and timeliness in three South African areas: a prospective study. BMC Public Health. 2011; 11(1): 404. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHaverkate M, D’Ancona F, Giambi C, et al.: Mandatory and recommended vaccination in the EU, Iceland and Norway: results of the VENICE 2010 survey on the ways of implementing national vaccination programmes. Euro Surveill. 2012; 17(22). pii: 20183. PubMed Abstract \| Publisher Full Text\n\nStock JH, Aspelund KM, Droste M, et al.: Estimates of the undetected rate among the SARS-CoV-2 infected using testing data from Iceland. medRxiv. 2020. Publisher Full Text\n\nDong L, Hu S, Gao J: Discovering drugs to treat coronavirus disease 2019 (COVID-19). Drug Discov Ther. 2020; 14(1): 58–60. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organization: Rational use of personal protective equipment for coronavirus disease (COVID-19): interim guidance, 19 March 2020. Technical report, World Health Organization, 2020. Reference Source\n\nLeung NHL, Chu DKW, Shiu EYC, et al.: Respiratory virus shedding in exhaled breath and efficacy of face masks. Nat Med. 2020. Publisher Full Text\n\nCowling BJ, Aiello AE: Public health measures to slow community spread of COVID-19. J Infect Dis. 2020; 221(11): 1749–1751. PubMed Abstract \| Publisher Full Text\n\nO’Neill LA, Netea MG: BCG-induced trained immunity: can it offer protection against COVID-19. Nat Rev Immunol. 2020; 20(6): 335–337. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLe Bert N, Tan AT, Kunasegaran K, et al.: SARS-CoV-2-specific T cell immunity in cases of COVID-19 and SARS, and uninfected controls. Nature. 2020; 584(7821): 457–462. PubMed Abstract \| Publisher Full Text\n\nPark SY, Kim YM, Yi S, et al.: Coronavirus Disease Outbreak in Call Center, South Korea. Emerg Infect Dis. 2020; 26(8): 1666–1670. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "74339", "date": "09 Nov 2020", "name": "Siddharth Sridhar", "expertise": [ "Reviewer Expertise emerging infectious diseases", "clinical virology", "epidemiology/ public health" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article is a description on some knowledge gaps surrounding asymptomatic COVID-19 burden. Good primer on herd immunity thresholds in the introduction. Some nice case studies. Reasonable discussion on the importance of asymptomatic/ pre-symptomatic transmission. However, there are some areas that require attention.\nOverall, this manuscript reads more like a commentary/ narrative review rather than an original 'research article'.\n\nMy specific criticisms are as follows:\n\nDefine variables in equation 1 below the equation.\n\nApart from R0, the concept of the effective reproduction number should also be introduced as a much more meaningful figure when NPIs are enforced.\n\nI recall closer to 700 cases on Diamond Princess. Please verify.\n\n“What is startling about these numbers is how few tested positive. Given the constrained environment of a cruise liner, a higher rate of social mixing would be expected than in normal living conditions.” This is somewhat simplistic. Once the outbreak started, you would expect significant reduction in social interactions and increasing mask usage/ isolation among passengers on board the Diamond Princess, which would lower the Rt.\n\nI do not understand how figure 2 and the equations relate to the rest of the paper. In fact, the first paragraph of the methods appears unrelated to the narrative case study/ review in the rest of the manuscript.\n\nI agree with the author on the importance of detecting asymptomatic shedders, but most current estimates of true asymptomatic COVID-19 rates are less than 50% (15 – 30% according to some meta-analyses). This needs to be updated. There is also a need to differentiate between true asymptomatic infection and ‘pre-symptomatic’ cases who are detected before eventually going on to detect symptoms.\n\nConsider citing Escobar LE et al1, PNAS as a peer-reviewed reference on the impact of BCG vaccination on severe COVID-19.\n\nBCG vaccination is unlikely to offer sterilizing immunity against COVID-19. Instead, it is mostly being investigated for its effect on ameliorating immune responses to prevent severe COVID-19. Therefore, we can’t rely on BCG vaccination to slow spread of COVID-19 and it is not a replacement for good-practice NPIs. Any country that practices high standards of NPIs, test-and-trace, and isolation-and-quarantine would be able to control COVID-19 irrespective of its BCG coverage. I feel the text is implying that BCG vaccination is under investigation for reducing COVID-19 community burden and this should be modified.\n\n“Another possible mitigating factor is that existing HIV remedies are among those being investigated for efficacy against COVID-19. However, it would be foolhardy to rely on this as a mitigating factor without solid evidence that it applies widely enough to matter.” May delete this point as lopinavir/ritonavir has been proven to be of no benefit in hospitalized COVID-19 patients in the WHO SOLIDARITY trial.\n\nFor the points in proposed research, several natural experiments looking at viral shedding in asymptomatic individuals (e.g. Han MS et al, Emerg Infect Dis2) and infectivity of asymptomatic cases (Gao M et al, Respir Med3) have already been described. Characterization of humoral and T-cell responses to COVID-19 have also been done in mild/ asymptomatic cases (Ko JH et al, J Clin Med4). The author may consider updating this section and reviewing the literature thoroughly to identify current research gaps.\n\nThe methods section in the abstract promised an examination of the strategy of using cloth face masks. However, there is no substantive discussion of cloth face masks in the text.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "6105", "date": "10 Nov 2020", "name": "Philip Machanick", "role": "Author Response", "response": "Thank you for constructive comments on the paper. Some of the the issues you raise are a consequence of trying to get this out quickly when this issue was fresh (version 1 was published on 4 May 2020). Unfortunately it has taken so long to find reviewers that some issues are out of date. I am happy to update the paper taking your comments into account. The asymptomatic vs. pre-symptomatic question is a good one. Since I wrote the paper increasing evidence has been published of viral shedding prior to symptoms showing. Your points on BCG are also good. As with several of your other points, missing some of this was a consequence of the long delay between submission and review. I will work through the other points along with any other reviews received. A thought experiment to clarify the role of R0 and Rt. R0 depends on 3 factors (slightly simplifying Delamater et al. 2019): inherent infectiousness of the pathogen how long an infected person remains contagious social mixing Assume R0 = 2.5 in a particular society (we must define this societally because of factor 3 – assuming susceptibility is uniform). While Rt is a current measure as opposed to R0, which is a base measure when everyone is susceptible, is it correct to say that NPIs change Rt and not R0? Imagine a particularly NPI is in effect when patient zero is imported. Then R0 will appear to be lower than without that NPI, because you have varied factor 3. However: if you wait until 40% of the population is infected and lift all NPIs, you will still expect to see herd immunity at 60% infected (based on R0 = 2.5). So an NPI fakes the effect of a disease with lower R0 though the measured effect is seen in Rt. Relax the NPI, and you put yourself onto a later part of the trajectory with a lower fraction susceptible than at the start, but Rt will now climb to the point it would have been without the NPI with that fraction (> 0) susceptible. So: I argue that even with NPIs in place, you really need to know R0, because that determines the inflection point at which cases decline if you relax your NPIs even if NPIs do not actually change R0. Otherwise you get the all-too-typical active-case curve that achieves a neat peak as if you reached herd immunity then takes off again when measures relax. There is a lot of confusion about R0 and Rt so it is good to get these ideas straight. I could add this to the paper if you consider this useful; otherwise it can stay here as a comment. Reference Delamater PL, Street EJ, Leslie TF, Yang YT, Jacobsen KH. Complexity of the Basic Reproduction Number (R0). Emerg Infect Dis. 2019 Jan;25(1):1-4. doi: 10.3201/eid2501.171901. PMID: 30560777; PMCID: PMC6302597." }, { "c_id": "6555", "date": "28 Apr 2021", "name": "Philip Machanick", "role": "Author Response", "response": "Define variables in equation 1 below the equation. Done – E is the only new variable and is now explicitly rather than implicitly defined. Apart from R0, the concept of the effective reproduction number should also be introduced as a much more meaningful figure when NPIs are enforced. Done – I explain the role of Rt in determining progress of a pandemic and referred to it as needed. I recall closer to 700 cases on Diamond Princess. Please verify. The number is from the reference I cite. I checked and cannot find one that contradicts this. “What is startling about these numbers is how few tested positive. Given the constrained environment of a cruise liner, a higher rate of social mixing would be expected than in normal living conditions.” This is somewhat simplistic. Once the outbreak started, you would expect significant reduction in social interactions and increasing mask usage/ isolation among passengers on board the Diamond Princess, which would lower the Rt. Reworded to reflect this. I do not understand how figure 2 and the equations relate to the rest of the paper. In fact, the first paragraph of the methods appears unrelated to the narrative case study/ review in the rest of the manuscript. I am trying to make the case that there is a phase of a pandemic where exponential growth will overtake health systems. Clarified. I agree with the author on the importance of detecting asymptomatic shedders, but most current estimates of true asymptomatic COVID-19 rates are less than 50% (15 – 30% according to some meta-analyses). This needs to be updated. There is also a need to differentiate between true asymptomatic infection and ‘pre-symptomatic’ cases who are detected before eventually going on to detect symptoms. I found meta-analyses supporting 15–20%, which I think establishes your point that the fraction could be significantly lower. I added also the potential confounder of miscounting pre-symptomatic as asymptomatic. Consider citing Escobar LE et al1, PNAS as a peer-reviewed reference on the impact of BCG vaccination on severe COVID-19. Done, thanks for the reference. BCG vaccination is unlikely to offer sterilizing immunity against COVID-19. Instead, it is mostly being investigated for its effect on ameliorating immune responses to prevent severe COVID-19. Therefore, we can’t rely on BCG vaccination to slow spread of COVID-19 and it is not a replacement for good-practice NPIs. Any country that practices high standards of NPIs, test-and-trace, and isolation-and-quarantine would be able to control COVID-19 irrespective of its BCG coverage. I feel the text is implying that BCG vaccination is under investigation for reducing COVID-19 community burden and this should be modified. Done – and added a caution based on hindsight that correlation ≠ causation, with the example of a similar study of ivermectin in Peru that does not appear to be holding up. “Another possible mitigating factor is that existing HIV remedies are among those being investigated for efficacy against COVID-19. However, it would be foolhardy to rely on this as a mitigating factor without solid evidence that it applies widely enough to matter.” May delete this point as lopinavir/ritonavir has been proven to be of no benefit in hospitalized COVID-19 patients in the WHO SOLIDARITY trial. A useful update – a reader who compares versions of the paper and your review will see this history so I am happy to delete this as requested. This illustrates the value of the open review model; the reader can see how our (not only – my) undestanding of the pandemic has evolved since the initial version of the paper. For the points in proposed research, several natural experiments looking at viral shedding in asymptomatic individuals (e.g. Han MS et al, Emerg Infect Dis2) and infectivity of asymptomatic cases (Gao M et al, Respir Med3) have already been described. Characterization of humoral and T-cell responses to COVID-19 have also been done in mild/ asymptomatic cases (Ko JH et al, J Clin Med4). The author may consider updating this section and reviewing the literature thoroughly to identify current research gaps. Thank you for the references. I have read the Gao et al. paper and it seemed to me methodologically weak and this is supported by a follow-up letter by Silverman et al.5. However you make a good point that I should review progress in this regard so I have added new references to this section and added the new gap of understanding how vaccines control infectiousness. Han et al. is interesting, thanks. That led me to finding a more recent more authoritative study, Sayampanathan et al.6 I found a good reference on T cell response, Sekine et al7. Thanks for pointing to the need to look into this further. The methods section in the abstract promised an examination of the strategy of using cloth face masks. However, there is no substantive discussion of cloth face masks in the text. I removed this and toned down the BCG vaccine claim in the abstract. The main focus of the paper is clarified as emphasising the value of understanding asymptomatic and mild cases – even though the immediate medical emergency with a novel disease is saving the most severe cases. References 1. Escobar L, Molina-Cruz A, Barillas-Mury C: BCG vaccine protection from severe coronavirus disease 2019 (COVID-19). Proceedings of the National Academy of Sciences. 2020; 117 (30): 17720-17726 2. Han M, Seong M, Kim N, Shin S, et al.: Viral RNA Load in Mildly Symptomatic and Asymptomatic Children with COVID-19, Seoul, South Korea. Emerging Infectious Diseases. 2020; 26 (10): 2497-2499 3. Gao M, Yang L, Chen X, Deng Y, et al.: A study on infectivity of asymptomatic SARS-CoV-2 carriers.Respir Med. 169: 106026 4. Ko JH, Joo EJ, Park SJ, Baek JY, et al.: Neutralizing Antibody Production in Asymptomatic and Mild COVID-19 Patients, in Comparison with Pneumonic COVID-19 Patients.J Clin Med. 2020; 9 (7). 5. Silverman, E. P., Reddy, R., Ataya, A., & Eagan, C. (2020). Letter to the Editor concerning A study on infectivity of asymptomatic severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) carriers by Gao et al. Respiratory Medicine, 106061. Advance online publication. https://doi.org/10.1016/j.rmed.2020.106061 6. Sayampanathan AA, Heng CS, Pin PH, Pang J, Leong TY, Lee VJ. Infectivity of asymptomatic versus symptomatic COVID-19. The Lancet. 2021 Jan 9;397(10269):93-4. 7. Sekine T, Perez-Potti A, Rivera-Ballesteros O, Strålin K, Gorin JB, Olsson A, Llewellyn-Lacey S, Kamal H, Bogdanovic G, Muschiol S, Wullimann DJ. Robust T cell immunity in convalescent individuals with asymptomatic or mild COVID-19. Cell. 2020 Oct 1;183(1):158-68." } ] }, { "id": "77927", "date": "09 Feb 2021", "name": "Agata Mikolajczyk", "expertise": [ "Reviewer Expertise microbiology", "molecular biology", "translational medicine" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAlthough author of this work arise few types of COIVD-19 course among lot of variety, it gives some important general view. Nevertheless there is no type where pandemic were managed very effectively. Only there is some discussion about Iceland giving an example of extensive testing but this could be support with showing strategy of other countries which managed the best the COVID - 19 like New Zeland or Taiwan and point what was the common for this three countries.\n\nBecause work was writing some time ago and situation during pandemic is changing very dynamically especially in the context of vaccines, some of given strategies how to managed with current pandemic can be not timely . However it has general value as strategy for coping with any pandemic so in my opinion it is important content of this work.\n\nThere is lack of any detailed data which correlate wearing mask with spread of virus. There is some discussion about this and references, but here could be some investigation like: comparing spread rate of virus in countries after obligated society to wearing mask always with the one which hadn’t obligate.\n\nAs I could see the report from second reviewer I will not double the point he has raised and I support his comments.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "6342", "date": "11 Feb 2021", "name": "Philip Machanick", "role": "Author Response", "response": "Thank you for your constructive comments and being willing to accept that an article that has taken a long time to get through review is worth updating for the current context. When I first wrote it, I hoped to get it through review fast to add to the debate about asymptomatic transmission. When I had no reviews on the first version, I updated it; doing so again, thanks to the f1000 model of keeping all version live, unintentionally turns it into a living review." }, { "c_id": "6556", "date": "28 Apr 2021", "name": "Philip Machanick", "role": "Author Response", "response": "Although author of this work arise few types of COIVD-19 course among lot of variety, it gives some important general view. Nevertheless there is no type where pandemic were managed very effectively. Only there is some discussion about Iceland giving an example of extensive testing but this could be support with showing strategy of other countries which managed the best the COVID - 19 like New Zeland or Taiwan and point what was the common for this three countries. Thank you for raising this point. Now that we have more history, New Zealand and Taiwan present an interesting contrast in how to eliminate community spread – respectively by locking down hard and early before being ready for the appopriate measures versus having the measures in place already1. I added this in to the case studies. Because work was writing some time ago and situation during pandemic is changing very dynamically especially in the context of vaccines, some of given strategies how to managed with current pandemic can be not timely . However it has general value as strategy for coping with any pandemic so in my opinion it is important content of this work. Thank you – I agree. As I was preparing my update, I heard the British Prime Minister, in response to a question on his biggest mistake, admitting that it was failing to consider asymptomatic transmission. There is lack of any detailed data which correlate wearing mask with spread of virus. There is some discussion about this and references, but here could be some investigation like: comparing spread rate of virus in countries after obligated society to wearing mask always with the one which hadn’t obligate. Since the first reviewer pointed out that coverage of masking was inadequate I decided to remove this as a major focus. I have attempted to look into this issue but there are too many confounders to arrive at a definitive conclusion (e.g., if mask-wearing reduces other NPIs in some societies, that would reduce the effectiveness of masks)2. In general terms the fact that asymptomatic transmission is a factor indicates that mask wearing is likely to be effective, but I have found no definitive study to back this – though e.g. the Czech experience of having one of the lowest rates of infection in Europe while masking and becoming one of the worst when masking was dropped supports the case. For this reason, I prefer to make the focus asymptomatic transmission and dropped masking as an issue. As I could see the report from second reviewer I will not double the point he has raised and I support his comments. Thank you – see my responses to the other reviewer. References 1. Summers J, Cheng HY, Lin HH, Barnard LT, Kvalsvig A, Wilson N, Baker MG. Potential lessons from the Taiwan and New Zealand health responses to the COVID-19 pandemic. The Lancet Regional Health-Western Pacific. 2020 Oct 21:100044. 2. Cartaud A, Quesque F, Coello Y. Wearing a face mask against Covid-19 results in a reduction of social distancing. Plos one. 2020 Dec 7;15(12):e0243023." } ] } ]	2	https://f1000research.com/articles/9-327
https://f1000research.com/articles/10-316/v1	23 Apr 21	{ "type": "Brief Report", "title": "Eye-related emergencies incidence at a tertiary referral center in Southern Italy during COVID-19 related lockdown", "authors": [ "Alfredo Niro", "Giancarlo Sborgia", "Rossella Favale", "Alessandra Sborgia", "Valentina Pastore", "Cristiana Iaculli", "Francesco Boscia", "Giovanni Alessio", "Giancarlo Sborgia", "Rossella Favale", "Alessandra Sborgia", "Valentina Pastore", "Cristiana Iaculli", "Francesco Boscia", "Giovanni Alessio" ], "abstract": "Background: In order to contain the community spread of coronavirus disease (COVID-19) in Italy, a stringent lockdown was imposed, which also impacted the healthcare services. The purpose of this study is to investigate the drop in the number of outpatients in the Ophthalmic Emergency Service (OES) in the Eye Clinic of University of Bari during the COVID-19 lockdown. Methods: A retrospective analysis of electronic medical records from a tertiary referral center in Southern Italy was performed. Demographics and medical characteristics of patients examined in the OES between March 10th, 2020 and May 3rd, 2020 were assessed and compared with records from the same period in 2019. We categorized the patients by sex, age group, and by the type of eye disease that was recorded as the principal diagnosis. The change (%) in the number of patients and diseases between the study periods was analyzed. Results: We observed a reduction in the number of OES visits during the lockdown (-63.4%) compared to the number of visits in 2019. The greatest changes were observed in the youngest (≤20 years; -76.6%) and the most elderly patients (≥81 years; -70.9%). The decrease in the number of patients presenting with each pathology ranged from -82% to -28.5%, depending on the pathology, and mainly involved the orbital and palpebral pathologies, anterior segment disorders, non-specific visual symptoms, and minor injuries. The diagnosis of chronic pathologies, rhegmatogenous retinal detachment, and vitreous haemorrhage increased by 134%, 100%, and 75%, respectively. Conclusions: Our results revealed the impact of the COVID-19 lockdown on OES activities in the Eye Clinic of University of Bari. A drop in number of visits across all age groups was observed. The number of patients presenting with minor and nonurgent conditions decreased, whereas the number of patients presenting with chronic diseases and urgent but deferrable conditions increased.", "keywords": [ "COVID-19", "Lockdown", "Ophthalmic Emergency Service", "Ocular pathologies" ], "content": "Introduction\n\nThe coronavirus disease (COVID-19), was first reported in China, on December 31st, 2019.1 The first case of COVID-19 in Italy was reported on February 21st, 2020.2 The World Health Organization (WHO) declared COVID-19 a pandemic on March 11th, 2020.3 The previous day, the Government of Italy had ordered a lockdown for a period of approximately two months. This involved introducing preventative measures such as closing schools and universities, halting non-essential industrial and commercial production activities, and limiting the movement of people throughout the country.4 As a result of the lockdown, all outpatient services were stopped, but emergency healthcare services continued to operate. A stringent system of triaging and screening of outpatients was introduced to mitigate the community spread of COVID-19.\n\nPrimary care practices reported reductions in the use of healthcare services due to the lockdown, including a reduction in the number of patients accessing surgical specialties, such as ophthalmology.5\n\nAround the world, eye-related complaints account for approximately 1–6% of the patients presenting to general emergency rooms.6 In the Eye Clinic of University of Bari, all ophthalmologists on staff work on a rotational shift pattern in the OES, which is open 24 hours a day, 365 days a year. Given the high volume of patients (over 70,000 a year; >200 patients a day), the hospital has a triaging system similar to that of a general hospital emergency room. In this study, we retrospectively conducted database searches of all patients presenting to our OES during the COVID-19 related lockdown in 2020 and compared them to records from the same period in 2019.\n\n\nMethods\n\nThe study setting was the Eye Clinic of University of Bari, a tertiary referral center in Southern Italy. Electronic records of OES patients from March 10th, 2020 (i.e., the day in which the quarantine measures were applied in Italy) to May 3rd, 2020 (i.e., the day in which the quarantine measures were discontinued) were searched in the central database of the Azienda Ospedaliero Universitaria Consorziale Policlinico di Bari, and compared with those of the same period the previous year (from March 10th, 2019 to May 3rd, 2019). The search was conducted by masking personally identifiable information, and selecting sex, age, and diagnosis as search parameters. Patients included in the study were categorized by age into the following groups: 0 to 20 years, 21 to 40 years, 41 to 60 years, 61 to 80 years, and 81 years or older. Only the principal diagnosis of each patient, based on the best judgment of the physician, was extrapolated from the central database for the analysis.\n\nIn order to summarize the broad spectrum of diseases observed during the study periods we identified 14 categories, including non-specific visual symptoms, orbital and palpebral pathologies, conjunctival pathologies, corneal pathologies, acute angle-closure glaucoma, posterior vitreous detachment, vitreous haemorrhage, rhegmatogenous retinal detachment, optic nerve pathologies, neurological pathologies, major injuries, minor injuries, ambulatorial pathologies, and post-surgical complications.\n\nThe change (%) in the number (n) of patients between the two study periods was calculated as follows: [(n 2019 − n 2020)/n 2019] ×100.\n\nThe patients involved in this study provided written informed consent for the publication of their clinical information. All the procedures were performed in agreement with the guidelines of the Declaration of Helsinki. The study was approved by the institutional review board of the Eye Clinic, Department of Medical Science, Neuroscience and Sense Organs, University of Bari on July 1st 2020.\n\n\nResults\n\nAll data that were accessed are available in Table 1. During the 2019 study period, there were 2387 instances of patients presenting to the OES (21% of all patients presenting to the clinic). During the 2020 study period, the number of patients presenting decreased to 872 (48% of all patients presenting to the clinic). In both study periods, more male patients accessed the OES than women, and the mean age category (61-80 years) remained the same for both study periods.\n\nIn both study periods, the proportion of visits by age category was similar, demonstrating that patients between 21 and 80 years old presented to OES more frequently than other age groups.\n\nThe percentage change in the number of children, adolescents, and young adults (age group, 0-20 years) presenting at the OES was the greatest (−76.6%) of all the age groups. This was followed by the elderly (age group, ≥81years) whose numbers reduced by −70.9% during the lockdown. The patient numbers in other age groups decreased by a range of −59% to −66.6% during the lockdown.\n\nConsidering the disorders affecting at least five patients in the study periods, the largest drop in number (−82%) was recorded in patients affected by conjunctival pathologies including bacterial and viral conjunctivitis, allergic conjunctivitis, and, rarely, granulomatous and neoplastic conjunctivitis. The percentage of patients complaining of orbital and palpebral pathologies and non-specific visual symptoms including blurred vision, reading difficulties, and metamorphopsia without a related pathology, had a considerable decrease (−68.8% and −67.3%, respectively).\n\nMinor injuries including chemical injuries or foreign bodies on the external eye decreased by −58% while major injuries including lid or corneal laceration and globe rupture decreased by only −28.5%. The percentage of post-surgical complications decreased by −53.4%.\n\nConversely, the number of patients with chronic diseases that are usually managed with outpatient follow-up (cataract, diabetic retinopathy, age-related macular degeneration, central serous chorioretinopathy, epiretinal membrane, glaucoma, uveitis, etc.) increased by 134%. In addition, the number of cases of vitreous haemorrhage and rhegmatogenous retinal detachment remarkably increased by 75% and 100%, respectively.\n\n\nDiscussion\n\nThis report aims to compare the number of patient accesses to an emergency department of a tertiary referral center, during the COVID-19 lockdown, compared to the same period in 2019.\n\nAs previously observed,7,8 there was a decreased number of OES visits across all age categories of the population during the lockdown. The number of patients presenting with acute diseases decreased, while the number of patients presenting with chronic and deferrable urgency conditions increased.\n\nAs previously reported,9 adults between 21 and 80 years old presented more frequently to the OES than any other age category of the population. However, during the lockdown, restrictions in mobility 3,4 and the fear of being infected with COVID-19 10 may have led to fewer OES visits.\n\nThe greatest percentage decrease in the number of patients presenting was in the vulnerable age groups of the population, i.e., the youngest and the elderly. The youngest people were limited in mobility because they carry a high viral load, meaning they are more contagious,11 and the older patients were limited in mobility because they are at a higher risk of severe illness from COVID-19.12\n\nAs was the case with previous studies,7,13 the most common eye conditions reported in the OES were conjunctivitis, ocular injuries, and palpebral pathologies. Within the 14 categories of diseases, the largest decrease was observed in the conjunctival disorders. It is likely that this is because acute conditions were managed at home, and people with non-acute conditions delayed their OES visit.\n\nThe reduction in patient numbers varied depending on the ocular injury, as is in line with previous reports.14–16 This could be explained by the change of the injury setting, i.e., the types and frequency of injuries typically occurring in workplaces, schools, and during social activities may differ to the those occurring at home during the lockdown.15,16\n\nDuring the lockdown, there was a considerable decrease in the percentage of patients complaining of non-specific visual symptoms without a related pathology, demonstrating that many patients that usually present to the OES, probably do not need emergency care.9\n\nThe percentage of post-surgical complications decreased due to the deferring of elective surgical procedures during the lockdown.17\n\nIt is likely that patients with chronic conditions, that are usually managed by outpatient follow-up, presented to the OES due to the closing of all non-essential outpatient activities during the lockdown. The increase in vitreous haemorrhage cases may be due to unbalanced hemostasis in vasculopathy patients taking oral anticoagulants, or the progression of retinal vascular pathologies as diabetic retinopathy. Due to the temporary suspension of surgical activities or the conversion of other nearby hospitals to COVID-19 centers, a large number of cases of rhegmatogenous retinal detachment may have been referred to our clinic as a tertiary referral center.\n\nA report on the health care services of the Commonwealth Area during the COVID-19 lockdown showed that surgical specialties such as ophthalmology were most affected by the drop of visits, with a -79% decrease in the volume.5 The reduction in the number of OES visits could be due to the limitations imposed by the government as well as the risk-stratification guidelines introduced by ophthalmic departments to limit and optimize the treatment and care of ocular disorders.18,19 Furthermore, some patients may have intentionally avoided medical care rather than risking coronavirus exposure at hospitals.10\n\nOn the other hand, the suspension of outpatient services may have led to the progression of some systemic 20–22 and ocular diseases 17 which, in turn, may have prompted patients to go to the emergency services.\n\nThe impact of the government’s acts, hospital risk-stratification guidelines, and population anxiety on OES activity during the COVID-19 lockdown could be an indicator of the short-term effects that the lockdown has on public health. To date, there is also uncertainty over the possibility of a reboot of the lockdown, due to a new increase of the contagion curve, despite the beginning of coronavirus vaccination campaign in Europe on December 27th, 2020. Therefore, an analysis of ophthalmic emergency service activities, such as the one presented here, may prove useful in defining new guidelines to reorganize and rationalize outpatient access to public health services.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "References\n\nHuang C, Wang Y, Li X, et al.: Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet. 2020; 395(10223): 497–506. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSpina S, Marrazzo F, Migliari M, et al.: The response of Milan's Emergency Medical System to the COVID-19 outbreak in Italy. Lancet. 2020; 395(10227): e49–e50. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAccessed 28th Apr 2020. Reference Source\n\nAccessed 29th Apr 2020. Reference Source\n\nMehrotra A, Chernew M, Linetsky D, et al.: The impact of the COVID-19 pandemic on outpatient visits: a rebound emerges. Accessed 12th Jul 2020. Reference Source\n\nJafari AK, Bozorgui S, Shahverdi N, et al.: Different causes of referral to ophthalmology emergency room. J Emerg Trauma Shock. 2012; 5(1): 16–22. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPellegrini M, Roda M, Lupardi E, et al.: The impact of COVID-19 pandemic on ophthalmological emergency department visits. Acta Ophthalmol. 2020; 98(8): e1058–e1059. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPosarelli C, Maglionico MN, Covello G, et al.: Ophthalmological emergencies and the SARS-CoV-2 outbreak. PLoS One. 2020; 15(10): e0239796. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChanna R, Zafar SN, Canner JK, et al.: Epidemiology of Eye-Related Emergency Department Visits. JAMA Ophthalmol. 2016; 134(3): 312–9. PubMed Abstract \| Publisher Full Text\n\nMata F: The Fears of Being Infected by the COVID–19 Virus in Canada: A Look At Germophobes, Crowd-Averse, Fearless and Other Population Segments. SocArXiv. Nov. 2020: 4. Publisher Full Text\n\nYonker LM, Neilan AM, Bartsch Y, et al.: Pediatric Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2): Clinical Presentation, Infectivity, and Immune Responses. J Pediatr. 2020; 227: 45–52.e5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhou F, Yu T, Du R, et al.: Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study. Lancet. 2020; 395(10229): 1054–1062. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKang EYC, Tai WC, Lin JY, et al.: Eye-related Emergency Department Visits with Ophthalmology Consultation in Taiwan: Visual Acuity as an Indicator of Ocular Emergency. Sci Rep. 2020; 10(1): 982. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStedman EN, Jefferis JM, Tan JH: Ocular Trauma During the COVID-19 Lockdown. Ophthalmic Epidemiol. 2021: 1–3. Epub ahead of print. PubMed Abstract \| Publisher Full Text\n\nWu C, Patel SN, Jenkins TL, et al.: Ocular trauma during COVID-19 stay-at-home orders: a comparative cohort study. Curr Opin Ophthalmol. 2020; 31(5): 423–426. PubMed Abstract \| Publisher Full Text\n\nPellegrini M, Roda M, Di Geronimo N, et al.: Changing trends of ocular trauma in the time of COVID-19 pandemic. Eye (Lond). 2020; 34(7): 1248–1250. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nToro MD, Brézin AP, Burdon M, et al.: Early impact of COVID-19 outbreak on eye care: Insights from EUROCOVCAT group. Eur J Ophthalmol. 2021; 31(1): 5–9. PubMed Abstract \| Publisher Full Text\n\nParravano M, Borrelli E, Costanzo E, et al.: Protect healthcare workers and patients from COVID-19: the experience of two tertiary ophthalmology care referral centers in Italy. Ophthalmol Ther. Jun. 2020; 9(2): 231–234. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBorrelli E, Sacconi R, Querques L, et al.: Taking the right measures to control COVID-19 in ophthalmology: the experience of a tertiary eye care referral center in Italy. Eye (Lond). 2020; 34(7): 1175–1176. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDanhieux K, Buffel V, Pairon A, et al.: The impact of COVID-19 on chronic care according to providers: a qualitative study among primary care practices in Belgium. BMC Fam Pract. 2020; 21(1): 255. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWright A, Salazar A, Mirica M, et al.: The Invisible Epidemic: Neglected Chronic Disease Management During COVID-19. J Gen Intern Med. 2020; 35(9): 2816–2817. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSaqib M, Siddiqui S, Qasim M, et al.: Effect of COVID-19 lockdown on patients with chronic diseases. Diabetes Metab Syndr. 2020; 14(6): 1621–1623. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "83823", "date": "17 May 2021", "name": "Grayson W Armstrong", "expertise": [ "Reviewer Expertise Ophthalmology", "epidemiology", "eye trauma" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present a study evaluating the changes in patient demographics and diagnoses during the lockdown in Italy at a single eye center at the University of Bari.\nThere are a few major revisions necessary in order to make the manuscript scientifically sound:\nYou must evaluate the statistical significance of any changes reported in the article. Proportions and percentages alone are insufficient.\n\nThe diagnostic groupings used seem to be created by the authors for this individual study. If these same diagnostic groupings were used prior, please cite that paper. Otherwise, they are inappropriate as written. The diagnostic groupings will have a major impact on the outcomes of the paper, but the reasoning behind the diagnostic groupings is not ever spelled out and is unclear. The diagnoses that account for each grouping are not listed for the reader and should be made transparent. Additionally, the listing of some individual diagnoses (vitreous hemorrhage, RRD, PVD, angle closure glaucoma) seem to be provided out of convenience as these showed major changes, while others are grouped into massive categories (ambulatorial pathologies). Lastly, no evaluation of urgent versus non-urgent diagnoses is provided, as some optic nerve pathologies are urgent (i.e. optic neuritis, papilledema), while others are not (optic nerve drusen). I would recommend using previously published diagnostic groupings or explain the rationale for the groups, as well as the diagnoses included in each group.\n\nPlease indicate whether source data would be available in a repository or publicly available. Currently, the percentages provided as not sufficient to ensure reproducibility.\nLess major revisions that should be included are as follows:\nIn the introduction, an effort should be made to cite all relevant literature that shows reductions in ophthalmic emergencies presenting to hospitals across the world. I have included four articles that should be included1,2,3,4, but this is not an exhaustive list. Please include these and others.\n\nThe final sentence of the introduction is better suited for the methods section than the introduction.\n\nThe methods section should clearly state that this is a retrospective cross sectional study. It should also clearly deliniate inclusion criteria (these are mentioned but not described as such) and exclusion criteria (if none, say so).\n\nWas there any evaluation for duplicate patients coming in multiple times for the same diagnosis?\n\nWhen you say 'best judgement of the physician', is this the treating physician or the physician reviewing the chart retrospectively?\n\nI believe the calculation you provided should say [(n 2020- n2019)/n2019]. Is that correct?\n\nIn the results section, you state that these patients represent a percentage of patients presenting to the clinic. Did the other patients present to the ambulatory clinic instead of the emergency dept? Make this clear.\n\nWhen you talk about male patients you should provide percentages and p-values.\n\nWhat do you mean by 'mean age category'? The category that contains the mean age of patients? Please explain.\n\nTable 1: Please calculate statistical significant for all values presented.\n\nWere there truly zero RRDs at your institution during the time period of 2019?\n\nIn the Discussion section, in first sentence, please change 'patient accesses to an' to 'patients who accessed the'.\n\nSome of the conclusions seem to be going beyond the data presented and should either be removed or the study changes to support the conclusions. Examples include: 'number of patients with acute diseases decreased' - you did not subcategorize all diagnoses based on acute versus chronic; 'chronic and deferrable urgency conditions increased' - same as above; 'acute conditions were managed at home' - this is not supported by your study similar to the above; 'people with non-acute conditions delayed their OES visit' - this goes against what you were saying above, and is further not supported by data; 'many patients that usually present to the OES, probably do not need emergency care' - this is not supported by the study, as it is unclear if the patients who avoided the emergency dept did not need to be seen.\n\nFor the comment on vitreous hemorrhage, did you evaluate whether these patients were on blood thinners or if they had diabetic retinopathy as a cause? This data is not presented, and therefore the assumption goes beyond the scope of the data presented.\n\nThe report of the 'Commonwealth Area' should instead refer to the United States. The Mehrotra et al. study is a US-wide survey published by the commonwealth fund.\n\nRemove reference to the potential reboot of the pandemic lockdown as this is speculatory.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? No\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "96055", "date": "26 Oct 2021", "name": "Roberto dell'Omo", "expertise": [ "Reviewer Expertise Medical and surgical retina" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAlfredo Niro et al. present a study evaluating the changes in patient demographics and diagnoses during a two-month lockdown period at a single eye center in the South of Italy. I suggest the following changes in order to improve the quality of the manuscript:\nThe authors should report the statistical significance of the data analysed in the article.\n\nThe centre of Bari was included in a large retrospective study including 39 referral centers in Italy and investigating the reduction of urgent and non-urgent surgical procedures carried out in the period of lockdown in comparison to the same period in 2019. The authors should clarify if there is an overlapping between the patients included in that study and the patients of this current study. Should also make a comment if their data are not in line with those observed in the rest of Italy.\n\nGenerally, papers comparing the surgical procedures between lockdown periods and control periods, have distinguished between urgent and elective procedures. Conversely the authors, chose to select 14 categories of diseases, the details of which are rather vague and questionable. For example it is not clear why optic nerve pathologies are kept separated from neurological pathologies. What are the criteria to judge an injury as minor or major? What the authors mean for “ambulatorial pathologies” etc?\n\nIn the methods section it should be clearly state that this was a retrospective cross sectional study.\n\nIn the methods section the authors propose to investigate several intervals of age: 0 to 20 years, 21 to 40 years, 41 to 60 years, 61 to 80 years, and 81 years or older. Could they explain why and on what basis these intervals were chosen?\n\nHow do the authors explain the fact that no cases of RRD presented to the emergency department in 2019?\n\nThe paper was written several months ago and from that time, the cases of COVID have decreased significantly making the chance of having a further lockdown in Italy very remote. The authors should change the conclusions of the manuscript accordingly.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/10-316
https://f1000research.com/articles/9-3/v1	06 Jan 20	{ "type": "Research Article", "title": "A bioactive compound isolated from Duku (Lansium domesticum Corr) fruit peels exhibits cytotoxicity against T47D cell line", "authors": [ "Khusnul Fadhilah", "Subagus Wahyuono", "Puji Astuti", "Khusnul Fadhilah", "Puji Astuti" ], "abstract": "Background: Breast cancer is a major health problem for women globally. Many attempts have been promoted to cure cancer by finding new anticancer medicines from natural resources. Despite the richness of biodiversity discovered, there are some natural resources that remain unexplored. Fruit peels of Duku (Lansium domesticum Corr.) are rich with compounds that may have the potential to be developed as anticancer drugs. This study aimed to isolate cytotoxic compounds from the fruit peels of L. domesticum and assess their cytotoxic nature against T47D cells. Methods: Powdered peels were macerated with ethyl acetate and the filtrate was evaporated to give EtOAc extract A. Dried extract A was triturated with n-hexane to give n-hexane soluble fraction B and insoluble fraction C. The cytotoxic nature of these three samples were assessed using MTT assay using T47D cells and doxorubicin as a control. Results: Fraction C that showed the smallest IC50 (25.56 ± 0.64μg/mL) value compared to extract A and fraction B. Fraction C was further fractionated by vacuum liquid chromatography to give 6 subfractions. Subfraction 2 showed a single compound based on thin layer chromatography, and this compound was identified as Lamesticumin A on the basis of its spectroscopic data. Lamesticumin A demonstrated cytotoxic activity against T47D cell lines with an IC50 value of 15.68 ± 0.30µg/mL. Conclusions: Further research is needed to investigate the potential of the natural compound Lamesticumin A derived from L. domesticum fruit peel as an anticancer therapy.", "keywords": [ "Lansium domesticum Corr.", "cytotoxic", "T47D cell line", "Lamesticumin A" ], "content": "Introduction\n\nThe most frequent cancer in women and that which causes the highest mortality is breast cancer. In Indonesia, it was reported that approximately 21% of cancer deaths among women were due to breast cancer1. Therefore, new medicines to eradicate this type of cancer is required. Duku (Lansium domesticum Correa) widely grows in Indonesia. Traditionally, L. domesticum bark and seeds have been used to treat dysentery and fever2. Based on previous studies, chloroform and methanol extracts of L. domesticum displayed cytotoxic activity on murine melanoma (B16F10) and colon cancer (HT29) cells3. In addition, it has been shown that ethanol and ethyl acetate fractions of the peel have a deterrent activity on DNA damage in lymphoblast cells induced by H2O2 exposure4. Onoceranoid-type of triterpenoids have been isolated from twigs and leaves of L. domesticum, and these compounds showed antibacterial and antimutagenic activities5,6. In this study, the cytotoxic effects of compound extracted from the peels of L. domesticum are assayed against breast cancer T47D cells.\n\n\nMethods\n\nThe fruits of L. domesticum were collected on March 2018 from Bantul, Yogyakarta (GPS : -7.871098, 110.394854) and identified at the Department of Pharmaceutical Biology, Faculty of Pharmacy, Universitas Gadjah Mada.\n\nOrganic solvents (methanol, ethyl acetate, chloroform, n-hexane) used were pro analytical grades obtained from Merck. Silica gel F254, Silica gel PF254, (Merck), RPMI 1640, Fetal Bovine Serum, Penicillin-Streptomycin, Fungizon, Sodium bicarbonate (Gibco), HEPES (Invitrogen), Phosphate Buffered Saline, MTT (Sigma Aldrich cat. M5655), Doxorubicin (Sigma Aldrich). Infrared (KBr) spectrum was obtained from spectrophotometer (Shimadzu) using a method previously described by Ashokkumar and Ramaswamy7. Ultraviolet spectrum (CHCl3) was obtained from UV spectrophotometer (Hitachi UH 5300). Sample (1 mg) were diluted in 1 mL CHCl3 and was run between 200–400 nm. Spectra of 1H- and 13C- NMR in CDCl3 solvent were measured using JEOL JNM-ECZ 500R/S1 at 500 MHz.\n\nThe peel was separated from the fruit, dried in oven 50°C for 24 hours and powdered using a blender. Powdered L. domesticum fruit peel (200 gr) was macerated with ethyl acetate (EtOAc; 2 L) overnight. This solution was filtrated (0,45μm) and the filtrate was evaporated to dryness with a rotary evaporator set at 50°C, to give dried EtOAc extract (A, 50.13 g). In order to separate the extract into non-polar and polar fractions, 6 gr of extract A was diluted 5 times using n-hexane (20 mL) to give soluble n-hexane fraction B (supernatant; 3.03 g) and insoluble n-hexane fraction C (residue; 2.83 g).\n\nFraction C was the most active among other fractions (see Results), and was therefore further fractionated using vacuum liquid chromatography as described by Mae Sri Hartati et al.8. In brief, using silica gel preparation grade (15 gr) as stationary phase this was eluted with n-hexane and increasing amounts of ethyl acetate. Six subfractions were obtained and subfraction 2 contained a major compound which appeared as white crystals (referred to as compound 1). Compound 1 was obtained as a single compound from subfraction 2, while the other subfractions still contained various compounds. Compound 1 (142.6 mg), had a melting point at 140–150°C (Figure 1). Compound 1 was identified using spectroscopy data such as ultraviolet (UV), infrared (IR), 13C-NMR and 1H-NMR (see section Chemicals and equipment).\n\nThe bioassay followed the methodology described by Bahuguna et al.9 with modifications. In brief, 100 μl T47D cells (in RPMI mediaFaculty of Medicine, Universitas Gadjah Mada) were placed in each well of a 96 well micr oplates, resulting in 1 × 104 cells/well. The cells were incubated for 24 hours at 37°C in a CO2 incubator.\n\nExtract A, fractions B and C and compound 1 (5mg) were dissolved in DMSO (50 μL). Serial concentrations of extract and fractions (50, 25, 12.5, 6.25, 3.125 μg/mL), compound 1 (25, 12.5, 6.25, 3.125 μg/mL) and doxorubicin (positive control; 0.5, 0.25, 0.125, 0.0625, 0.0312 μg/mL) were obtained. Cells were treated with the dose dependent samples and incubated for 24 hours at 37°C. The culture medium was removed by pipette, and MTT solution (100μL) was added to each well and incubated for 4 hours at 37°C. After incubation, stop solution (10% SDS, 100 μL) was added to each well and let stand at room temperature for 24 hours.\n\nAbsorbance was measured by microplate reader (Bio Rad) at 595 nm. positive control The data generated were used to plot a dose-response curve and IC50 of the samples was determined.\n\nThe IC50 values were analyzed by one-way ANOVA with statistical significance P < 0.05 using IBM SPSS ver.23.\n\n\nResults\n\nIdentified as Lamesticumin A.\n\nWhite crystal. IR (KBr) vmax cm-1: 3074, 2960, 1712; UV (MeOH)λmax 236,5; 1H,13C-NMR: see Table 1; m/z 502; (Calculated for C31H50O5)\n\n1H- and 13C- NMR (CDCl3) spectra were obtained from JEOL JNM-ECZ 500R/S1, 500 MHz\n\nThe infrared spectroscopy (KBr) spectrum of 1 showed a broad band at 3400–2800 cm-1, which indicated the presence of –OH group, specifically –COOH due to intermolecular bonding. This data is supported by the appearance of –C=O at 1712 cm-1. Compound 1 displayed UV absorption at 236,5 nm. The 13C-NMR spectrum (500 MHz, CDCl3) of compound 1 showed 30 carbons (Table 1). There were two down field carbon signals (δ, 147.6 and 148.1 ppm) identified as C=O signal carbons. Two characteristic terminals =CH2 signals (δ, 107.4 and 114.2 ppm) were observed, and this identity was confirmed by 2D (Het-Cor) NMR technique. Based on 13C-NMR and 1H-NMR data, compound 1 (Figure 2) was identified as Lamesticumin A (C31H50O5, m/z, 502) which was previously isolated from L. domesticum twigs5.\n\nThe cytotoxicity of extract A, fractions B and C, compound 1 and doxorubicin (positive control) is shown in Table 2. Fraction C was the most cytotoxic (IC50 25.57 μg/mL) compared with extract A (29.41 μg/mL) and fraction B (43.51 μg/mL). The IC50 of the isolated compound from fraction C, compound 1/Lamesticumin A was 15.68 μg/mL. All samples inhibited T47D cell growth in a dose dependent behavior (Figure 3).\n\nError bars shows standard deviation.\n\n\nDiscussion\n\nIn this study, the cytotoxic activity of Lamesticumin A, derived from the peel of L. domesticum, was demonstrated in the T47D cell line with IC50 15.68 (μg/ml). The T47D cell line is an epithelial breast cancer cell subtype luminal A cell line that express estrogen and progesterone receptors10. Based on National Cancer Institute guidelines, a natural compound has potent anticancer activity if it has IC50 <4 μg/ml or 10 μM11.\n\nMany triterpenoid compounds have been previously isolated from L. domesticum. Most of these compounds are UV inactive or have no strong UV absorbance because triterpenoid’s lack of a conjugated functional group12. Lansiosida A and Dukunolida A has been isolated from n-hexane extract of L. domesticum fruit peel13,14. Lamesticumin A is an onoceranoid-type triterpenoid, isolated previously from L. domesticum twigs, that has antibacterial activity against Staphylococcus aureus, Staphylococcus epidermidis, Micrococcus luteus, Bacillus subtilis, Micrococcus pyogenes and Bacillus cereus with minimum inhibitory concentration of <15 μg/ml5. Another onoceranoid-type triterpenoid Lansium acid I-IX were isolated from L. domesticum leaves, which was reported to have antimutagenic activity6.\n\nBased on several in vitro tests, some terpenoid compounds had anticancer activity. Sesquiterpene lactone compounds are known to inhibit Nf-kB, thereby inducing apoptosis15. Celastrol has anticancer properties by regulating various transcription factors, angiogenesis processes, cell cycle arrest and induction of apoptosis16. Betulinic acid can induce apoptosis in HT-29 colon cancer cells and acts as a chemosensitizer for chemotherapeutic agents in wildtype adenocarcinoma cancer cells (SNU-C5/WT)17. Clematangoticosides D and F from Clematis tangutica are known to have cytotoxic activity against human gastric cancer cell line (SGC-7901) with IC50 24.22 and 21.35 μM, respectively18. Cycloartane-type and oleanane-type triterpenoids from Ligularia przewalskii show cytotoxicity in Hela, HEPG2, SGC7901, MDA231, HL-60, and Lewis cell lines with IC50 8.40–24.39 μM19.\n\nIt has been reported that natural compounds combined with low doses of antineoplastics can increase effectiveness and reduce toxic effects20. Betulinic acid can induce apoptosis when combined with 5-fluorouracil, irinotecan and oxaliplatin4. Ursolic acid (UA), a pentacyclic triterpenoid, is known to have anticancer activity through interfering with multiple signaling pathways. Furthermore, UA has been shown to act as a chemosensitizing agent to increase the effect of conventional anticancer drugs21, and to increase the effect of doxorubicin by increasing the cellular amount of the drug in the MCF cell line22. Further study is needed to investigate the possibility of Lamesticumin A to be combined with doxorubicin for its potential to have synergistic effect.\n\n\nConclusions\n\nExtract, fractions and Lamesticumin A derived from the peel of L. domesticum showed cytotoxic activity against the T47D breast cancer cell line. Further research is needed to investigate the potential of the natural compound Lamesticumin A derived from L. domesticum fruit peel as an anticancer therapy.\n\n\nData availability\n\nZenodo: A bioactive compound isolated from Duku (Lansium domesticum Corr) fruit peels exhibits cytotoxicity against T47D cell line, http://doi.org/10.5281/zenodo.353967023.\n\nThis project contains the following underlying data:\n\n- UV, infrared, 13C-NMR and 1H-NMR spectra of compound 1.\n\n- Cell viability and IC50 values of extract A, fractions B and C, compound 1 and doxorubicin in T47D cell line.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nWHO: Cancer country profiles 2014. 2014; (Accessed: 20th April 2018). Reference Source\n\nHeyne K: Tumbuhan berguna Indonesia. Badan Litbang Kehutanan, 1988. Reference Source\n\nManosroi A, Jantrawut P, Sainakham M, et al.: Anticancer activities of the extract from Longkong (Lansium domesticum) young fruits. Pharm Biol. 2012; 50(11): 1397–407. PubMed Abstract \| Publisher Full Text\n\nKlungsupya P, Suthepakul N, Muangman T, et al.: Determination of Free Radical Scavenging, Antioxidative DNA Damage Activities and Phytochemical Components of Active Fractions from Lansium domesticum Corr. Fruit. Nutrients. 2015; 7(8): 6852–73. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDong SH, Zhang CR, Dong L, et al.: Onoceranoid-type triterpenoids from Lansium domesticum. J Nat Prod. 2011; 74(5): 1042–1048. PubMed Abstract \| Publisher Full Text\n\nMatsumoto T, Kitagawa T, Teo S, et al.: Structures and Antimutagenic Effects of Onoceranoid-Type Triterpenoids from the Leaves of Lansium domesticum. J Nat Prod. 2018; 81(10): 2187–2194. PubMed Abstract \| Publisher Full Text\n\nAshok kumar R, Ramaswamy M: Phytochemical screening by FTIR spectroscopic analysis of leaf extracts of selected Indian Medicinal plants. Int J Curr Microbiol App Sci. 2014; 3(1): 395–406. Reference Source\n\nMae Sri Hartati W, Mubarika S, Gandjar IG, et al.: Phalerin, a new benzophenoic glucoside isolated from the methanolic extract of Mahkota Dewa [Phaleria macrocarpa (Scheff). Boerl] leaves. Maj Farm Indones. 2005; 16(1): 51–57. Reference Source\n\nBahuguna A, Khan I, Bajpai VK, et al.: MTT assay to evaluate the cytotoxic potential of a drug. Bangladesh J Pharmacol. 2017; 12: 115–118. Reference Source\n\nYu S, Kim T, Yoo KH, et al.: The T47D cell line is an ideal experimental model to elucidate the progesterone-specific effects of a luminal A subtype of breast cancer. Biochem Biophys Res Commun. 2017; 486(3): 752–758. PubMed Abstract \| Publisher Full Text\n\nKuete V, Ndontsa BL, Nguekeu YMM, et al.: Ardisinol III, a naturally occurring alkenylmethylresorcinol displayed cytotoxic effects in carcinoma cells. Investig Med Chem Pharmacol. 2018; 1: 1–6. Reference Source\n\nXu R, Ye Y, Zhao W: Introduction to natural products chemistry.2011. Reference Source\n\nNishizawa M, Nademoto Y, Sastrapradja S, et al.: Structure of dukunolide A: A tetranortriterpenoid with a new carbon skeleton from lansium domesticum. J Chem Soc Chem Commun. 1985; 395–396.Publisher Full Text\n\nNishizawa M, Nishide H, Hayashi Y, et al.: The structure of Lansioside A : A novel triterpene glycoside with amino-sugar from lansium domesticum. Tetrahedron Lett. 1982; 23(13): 1349–1350. Publisher Full Text\n\nSharma SH, Thulasingam S, Nagarajan S: Terpenoids as anti-colon cancer agents - A comprehensive review on its mechanistic perspectives. Eur J Pharmacol. 2017; 795: 169–178. PubMed Abstract \| Publisher Full Text\n\nKashyap D, Sharma A, Tuli HS, et al.: Molecular targets of celastrol in cancer: Recent trends and advancements. Crit Rev Oncol Hematol. 2018; 128: 70–81. PubMed Abstract \| Publisher Full Text\n\nRzeski W, Stepulak A, Szymański M, et al.: Betulinic acid decreases expression of bcl-2 and cyclin D1, inhibits proliferation, migration and induces apoptosis in cancer cells. Naunyn Schmiedebergs Arch Pharmacol. 2006; 374(1): 11–20. PubMed Abstract \| Publisher Full Text\n\nZhao M, Da-Wa ZM, Guo DL, et al.: Cytotoxic triterpenoid saponins from Clematis tangutica. Phytochemistry. 2016; 130: 228–37. PubMed Abstract \| Publisher Full Text\n\nShi ZN, Wang YD, Gong Y, et al.: New triterpenoid saponins with cytotoxic activities from Ligularia przewalskii. Phytochem Lett. 2019; 30: 215–219. Publisher Full Text\n\nLagoa R, Silva J, Rodrigues JR, et al.: Advances in phytochemical delivery systems for improved anticancer activity. Biotechnol Adv. 2019; pii: S0734-9750(19)30063-1. PubMed Abstract \| Publisher Full Text\n\nPrasad S, Tyagi AK, Aggarwal BB: Chemosensitization by Ursolic Acid: A New Avenue for Cancer Therapy. In Role of Nutraceuticals in Chemoresistance to Cancer. 2018; 2: 99–109. Publisher Full Text\n\nZong L, Cheng G, Liu S, et al.: Reversal of multidrug resistance in breast cancer cells by a combination of ursolic acid with doxorubicin. J Pharm Biomed Anal. 2019; 165: 268–275. PubMed Abstract \| Publisher Full Text\n\nFadhilah K, Wahyuono S, Astuti P: A bioactive compound isolated from Duku (Lansium domesticum Corr) fruit peels exhibits cytotoxicity against T47D cell line [Data set]. Zenodo. 2019. http://www.doi.org/10.5281/zenodo.3539670" }	[ { "id": "70005", "date": "26 Aug 2020", "name": "Ratana Banjerdpongchai", "expertise": [ "Reviewer Expertise Programmed cell death", "Natural products", "Antioxidants", "Cancer." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMajor points: The extracts and purified bioactive compound, Lamesticumin A, from this plant, is not novel, since it was isolated from the twigs from this same herb in the previous report. The anticancer activity of the active compound is not promising since it is more than 4 microgram/mL or 10 micromolar, the level of which the National Cancer Institution defines is 4 microgram/mL or less than this. However, the authors discussed of using this compound as a chemosensitizing agent, which various steps still are needed before launching it as a therapeutic anti-cancer drug. The authors should explore the cytotoxic effects of the extracts, EtOAc extract (A), N-hexane fraction (B), N-hexane insoluble fraction (C) and compound 1 (Lamesticumin A) on normal cells.\nIn Figure 3, the percent cell viabilities of EtOAc extract (A), N-hexane fraction (B), N-hexane insoluble fraction (C) and compound 1 (Lamesticumin A) should be presented with the statistical significance by using asterisks, such as p<0.05, *p<0.01, and so on, above the dots.\nMinor points: The sources of chemicals and instruments should indicate the city and country that they are produced from. Sometimes the authors used gr and sometimes g, it should be consistent with each other. In subtopic “Cytotoxic assay”, in 3rd – 4th lines, the authors should rewrite the sentences. There are many grammar and various typological errors, such as the number of g it should be 43.4 rather than 43,4, etc. For the previous findings, the sentences should be in present tense, but for the authors’ research data in the Results and Discussion parts, the sentences would be in past tense. For MDA231, it should be MDA-MB-231 breast cancer cells. For MCF, it should be clarified as MCF-7 or not? In Ref. No. 12, there is no information of company, city, country of publication, is it a book? Ref. No. 21, the name of journal is full name, whereas others are in abbreviated forms. It should be consistent with each other.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6264", "date": "23 Apr 2021", "name": "Khusnul Fadhilah", "role": "Author Response", "response": "In the revised version, we present cytotoxic data on normal cells (Vero cell line) in Table 2 and Figure 3 has been revised as recommended by the reviewer for major point revision. Besides, we also present the minor points revision such as grammar and typological errors corrections." } ] }, { "id": "64744", "date": "17 Sep 2020", "name": "Deden Derajat Matra", "expertise": [ "Reviewer Expertise Agricultural Plant Science", "Horticulture", "AgronomyPhysiology", "Bioinformatics", "Molecular Biology." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe author did not describe the maturity degree from samples because the different stages of maturity may change secondary metabolites that concern effect to the bioactive compound.\nHow to handling samples after harvesting is important too. Please, author, describe more?\n\nIn conclusion, what kind of peel fresh or processed is best consumed as the higher potential to anticancer therapy.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-3
https://f1000research.com/articles/10-311/v1	22 Apr 21	{ "type": "Research Article", "title": "Thoughts on the potential to compensate a hearing loss in noise", "authors": [ "Marc René Schädler" ], "abstract": "Background: The effect of hearing impairment on speech perception was described by Plomp (1978) as a sum of a loss of class A, due to signal attenuation, and a loss of class D, due to signal distortion. While a loss of class A can be compensated by linear amplification, a loss of class D, which severely limits the benefit of hearing aids in noisy listening conditions, cannot. The hearing loss of class D is assumed to be the main reason why not few users of hearing aids keep complaining about the limited benefit of their devices in noisy environments. Working compensation strategies against it are unknown. Methods: Recently, in an approach to model human speech recognition by means of a re-purposed automatic speech recognition (ASR) system, the loss of class D was explained by introducing a level uncertainty which reduces the individual accuracy of spectro-temporal signal levels. Based on this finding, an implementation of a patented dynamic range manipulation scheme (PLATT) is proposed which aims to mitigate the effect of increased level uncertainty on speech recognition in noise by expanding spectral modulation patterns in the range of 2 to 4 ERB. This compensation approach is objectively evaluated regarding the benefit in speech recognition thresholds in noise using the ASR-based speech recognition model. Recommendations for an evaluation with human listeners are derived. Results: The objective evaluation suggests that approximately half of the class D loss due to an increased level uncertainty might be compensable. To measure the effect with human listeners, an experiment needs to be carefully designed to prevent the confusion class A and D loss compensations. Conclusions: A working compensation strategy for the class D loss could provide previously unexploited potential for relief. Evidence has to be provided in experiments with human listeners.", "keywords": [ "theoretical audiology", "speech perception modeling", "impaired hearing", "hearing loss compensation" ], "content": "Introduction\n\nTo this day, hearing aids without directional amplification or directional noise suppression provide their users only with limited benefit in noisy listening conditions. The limited benefit of hearing aids in noisy listening conditions is long known and was extensively described and put into context by Plomp (1978). There, the effect of impaired hearing on speech recognition performance was described as a sum of two fundamentally different classes of hearing loss: class A, which accounts for an attenuation of the signal, and class D, which accounts for a distortion of the signal. While the class A loss is defined such that it can be fully compensated by a suitable linear amplification of the signal, the class D loss was assumed to be level independent, that is, it cannot be compensated by linear amplification.\n\nThe class A loss, according to Plomp (1978), usually makes up the largest part of the total increase in speech recognition thresholds (SRTs) in quiet (A+D), which is why hearing aids provide the largest benefits in quiet environments. In noisy environments with sufficiently high levels, the contribution of the class A loss diminishes, and the contribution of the class D loss dominates the total loss. Plomp (1978) estimated that, on average, the class A loss accounts for approximately two-thirds and the class D loss for approximately one-third of the total loss, where huge individual variability was expected. A main drawback of this model is that it does not provide a specific hint on if and how the class D loss can be compensated.\n\nRecently, Kollmeier et al. (2016) proposed a modification to the feature extraction stage of the simulation framework for auditory discrimination experiments (FADE, Schädler et al., 2016a) to implement a class A loss as an absolute hearing threshold and a class D loss as a level uncertainty. The simulation approach with FADE employs a re-purposed automatic speech recognition system (ASR) to predict the outcome of a speech recognition test, such as the matrix sentence test (Kollmeier et al., 2015). The FADE approach was already successfully used to predict the outcomes of several speech in noise recognition experiments (Schädler et al., 2015, 2016b) as well as the outcomes of basic psycho-acoustic experiments (Schädler et al., 2016a) for listeners with normal hearing. Kollmeier et al. (2016) proposed to remove the information that is not available to an individual listener with impaired hearing in the feature extraction stage of the ASR system which is used in the FADE modeling approach.\n\nTo induce a class A loss in the model, variations in the internal spectro-temporal signal levels below the individual hearing threshold, determined by the individual audiogram, were removed. This manipulation is illustrated in the center panel of Figure 1, where the low-energy portions (blue/green) were replaced by constant values which are equal to the individual absolute hearing threshold, while the high-energy portions above the individual absolute hearing threshold are unchanged compared to unmodified representation in the upper panel. It is plausible that, if all relevant signal portions are above the hearing threshold, this manipulation should have no effect on the predicted speech recognition performance.\n\nFigure reproduced from Kollmeier et al. (2016) under the terms of the Creative Commons Attribution-NonCommercial 3.0 License.\n\nTo induce a class D loss in the model, random values were drawn from a normal distribution and added to the internal spectro-temportal signal levels, where the standard deviation of the normal distribution was a variable called level uncertainty. This manipulation is illustrated in the lower panel in Figure 1, where all signal portions, including those above the hearing threshold, are affected. Because the signal energy in that representation (which is a logarithmically scaled Mel-spectrogram) is represented in a logarithmic domain, linear amplification cannot be expected to change its effect on the predicted speech recognition performance.\n\nKollmeier et al. (2016) evaluated the effect of these manipulations on the predicted outcomes of the German matrix sentence test in a stationary and a fluctuating noise condition for different noise levels, and fitted the A/D-class description proposed by Plomp (1978) to the data. The results, reproduced in Figure 2, clearly show that the two manipulations largely achieved the intended effects, that is, inducing a class A and a class D hearing loss. The left panel shows FADE simulations with different standard audiograms from Bisgaard et al. (2010), which converge for high noise levels, that is, the manipulations can be compensated by amplification as one would expect from a class A loss. The right panel shows FADE simulations with increasing values for the level uncertainty, which do not converge for high noise levels, that is, the manipulations cannot be compensated by amplification as one would expect from a class D loss. An important observation of Kollmeier et al. (2016) was, that their empirical data set, which included matrix sentence test results in noise of almost 200 ears, could not be satisfactorily predicted with the class A loss alone, indicating that an implementation of a mechanism that induces a class D loss was needed to explain the speech recognition performance of individual listeners.\n\nThe left panel shows simulations with different absolute hearing threshold, which induce different class A losses. The right panel shows simulations with different values for the level uncertainty, which induce different class D losses. The embedded tables show the contributions of A and D when the descriptive model of Plomp (1978) was fitted to the data. Figure reproduced from Kollmeier et al. (2016) under the terms of the Creative Commons Attribution-NonCommercial 3.0 License. For further details please refer to the original publication.\n\nSchädler et al. (2020a) extended that approach by inferring the individual frequency-dependent level uncertainty from tone in noise detection thresholds, and achieved unprecedented accuracy in the prediction of benefits in SRTs due to different traditional hearing loss compensation schemes in noise and in quiet. The central assumption there is that the same mechanism that affects individual speech in noise perception also affects individual tone in noise perception. Other mechanisms that reduce the information encoded in the internal signal representation, like a reduced spectral resolution, might also be considered in this modeling approach. However, Hülsmeier et al. (2020) found that, with FADE, a reduced spectral resolution had only little effect on the simulated SRTs compared to an increased level uncertainty. This indicates that between a reduced spectral resolution and the level uncertainty, the latter is considered to be the suitable mechanism to implement a class D loss in FADE.\n\nIf a mechanism that removes the information in the auditory system of listeners with impaired hearing works similar to the level uncertainty, than the level uncertainty can be seen as the functional counter-part to a compensation strategy for a class D hearing loss. This is exactly what will be assumed in the remainder of this contribution to what can be considered theoretical audiology. In the context of FADE, this will allow to generate testable hypothesis on the achievable benefit in speech recognition performance due to a possible compensation strategy for a class D loss. The aim of this contribution is to:\n\nA) Present an approach which is able to partially compensate a class D loss as implemented with the level uncertainty in FADE, and\n\nB) objectively evaluate this approach and come up with testable quantitative hypothesis on its benefit in noisy listening conditions.\n\nFor an effective mitigation of the effect of the level uncertainty on speech recognition performance, three main problems need to be addressed:\n\n1) Which portions, or more precisely, patterns of the speech signal carry the most relevant information and need to be protected?\n\n2) Which signal patterns can be protected, given the strict constrains on the available future temporal context of the signal (approximately 1 ms) in hearing aid applications?\n\n3) How can such a protection be achieved?\n\nContinuing to follow an (assumed) analogy of basic principles in human and machine speech recognition, literature on robust automatic speech recognition provides hints on which signal patterns may be relevant for good (automatic) speech recognition performance in noise. Let us assume that a log Mel-spectrogram, such as it used for the calculation of the widely used Mel-frequency cepstral coefficient (MFCC) features, is representative for the information that is available to an ASR system. The spectral resolution of such a log Mel-spectrogram, of which an example is depicted in the upper panel of Figure 1, is about 1 equivalent rectangular bandwidth (ERB), the temporal resolution is 10 ms. The relevant speech information is encoded in the represented spectro-temporal dynamic, that is, the differences of spectro-temporal signal levels over time, called temporal modulations, and over frequency, called spectral modulations. It is remarkable that ASR systems traditionally don’t even use the whole information in the log Mel-spectrogram, but work with a reduced spectral resolution compared to the spectral resolution of the human auditory system. For example, the standard features used for ASR, MFCCs (ETSI, 2007), specifically encode spectral modulation frequencies from 0 to about 623 cycles per ERB; spectral modulation frequencies above 14 cycles per ERB empirically don’t contribute to automatic speech recognition performance. In line with this finding, the robust Gabor filter bank features use only slightly more than half of the available spectral resolution of the log Mel-spectrograms (c.f. Schädler et al., 2012). By omitting the corresponding parts of the feature vector, Schädler et al. (2012) assessed the relative importance of different spectro-temporal modulation frequencies in a robust ASR task. There, it was observed that the highest spectral modulation frequencies, around 14 cycles per band (the bandwidth of a Mel band there is approximately 1 ERB), seem to be important for the considered speech in noise recognition task. Before going into more detail on the importance of spectro-temporal modulations for noisy speech recognition, the limited possibilities to manipulate these in the context of hearing aids have to be considered.\n\nThe restriction to the availability of approximately 1 ms future temporal context in hearing aids does not allow for the reliable manipulation of temporal modulations below approximately 500 Hz. This limit is still way above the temporal modulation frequencies that are represented in the features of ASR systems. With the common analysis window length and shift of 25 and 10 ms, respectively, as used in Figure 1, the upper limit for represented temporal modulation frequencies is around 50 Hz. Hence, the only modulations that can be reliably manipulated are spectral modulations, which requires a temporal signal context in the order of 1 ms. Fortunately, spectral modulations play an important role in (automatic) speech recognition. With the already mentioned approach to model human speech recognition performance by means of a re-purposed ASR system, Schädler et al. (2016a) found that explicitly encoding spectral modulations, that is, across-frequency interactions, in the feature vector was required to explain the empirically found benefit when listening in a fluctuation masker. In other words, spectral modulations seem to be especially relevant in fluctuating noise maskers.\n\nIn combination of the answers to problems 1) and 2): spectral modulations just below 0.25 cycles per ERB, let’s say spectral patterns between 2 and 4 ERB, seem to be a good first candidate for protecting them against the level uncertainty. How can these signal patterns be protected or at least hardened against the effect of the internal noise due to the level uncertainty? In the logarithmic level domain (in the log Mel-spectrogram) the noise of the level uncertainty is additive, and one effective way of mitigating the effect of an additive noise is amplification. This means, that the desired spectral modulation patterns should be amplified, that is, expanded before the noise of the level uncertainty can remove that information.\n\nAt a first glance, an expansion of signal dynamic in the context of the reduced residual dynamic range of listeners with impaired hearing might seem completely undesirable: the expansion of spectral modulations can result in uncomfortably high and/or inaudibly soft signal levels; which can occur at the same time in different frequency ranges. However, considering the scale (2 to 4 ERB) it is clear, that this is a region which is not modified by common approaches to multi-band dynamic compression, where the signal is usually independently compressed in approximately six bands. The traditional approaches to multi-band dynamic compression with less than 9 bands leave spectral patterns with a width of 2 to 4 ERB virtually uncompressed, which might also be interpreted as an indication towards the importance of these patterns. If now the expansion of one (small, as we will later see) part of the spectral dynamic increases the total spectral dynamic range of the signal, it might be possible to counter it with a stronger compression of other (less relevant) parts of the spectral dynamic. However, compression should only be applied if it is required, that is, if the input signal dynamic range does not fit into the available output dynamic range. The dynamic range of speech in noise is less than the dynamic range of a clean speech signal. Especially when considering speech in only slightly fluctuating noises at signal-to-noise ratios (SNRs) around 0 dB, the dynamic range of the mixture can be close to the minimum which is required to discriminate words. In this condition, no improvement can be expected from compressing the signal dynamic. Hence, a micro-management of the limited residual dynamic range to optimize its use for speech-recognition-relevant information is a desirable goal. PLATT is an approach which gives preference to spectral modulation patterns that are supposedly relevant for speech recognition at the expense of spectral modulation patterns which are supposedly less relevant when mapping an input signal dynamic to a given output signal dynamic. The here outlined ideas are part of a German patent (DE 10 2017 216 972 B4); a patent application in Europe is pending.\n\nThe implementation details of PLATT, which are presented in the Methods, were already engineered towards low-delay real-time processing. In a simpler implementation, the representation of the log Mel-spectrogram could be manipulated in the MFCC domain, where the spectral modulations are suitably encoded for this operation, and which in MFCC-terminology would be called liftering. The inverse transform of the modified MFCCs, with amplified spectral modulation patterns between 0.25 and 0.125 cycles per band, could be compared to the unmodified log Mel-spectrogram, and a suitable filter response in the time-domain could be designed for each signal frame. While such an approach would have the advantage of simplicity (even simpler than the related implementation of an intelligibility-improving signal processing approach (IISPA) Schädler (2020b)), it would not be applicable without further modifications in a hearing device: The main problem would be the infeasible window length of 25 ms. Because of the highly non-linear nature of the interactions between the factors influencing speech recognition performance (speech material, masker type and level, reverberation, non-linear signal processing, hearing impairment), an implementation that already fulfills the most basic requirements of a hearing aid algorithm and can run on a hearing aid prototype1 was preferred over a simple proof-of-concept implementation. This additional effort makes a seamless translation to an application in a hearing device more likely and increases the meaningfulness of the presented results for a possibly realizable hearing aid solution.\n\nFor an evaluation of the implementation with respect to a possible compensation of a class D loss, the following points need to be considered:\n\n1) Which listening conditions, that is, which speech test and maskers, are suitable to evaluate the PLATT implementation objectively with FADE and (also later) empirically.\n\n2) Which listener profiles are suited to clearly demonstrate a (partial) compensation of a class D loss like it is implemented in FADE.\n\nThe first point is important to enable the verification with empirical data of any hypotheses that are based on the model predictions. Schädler et al. (2020a) discussed this point and proposed to use the SRT-50 measured with the matrix sentence test in quiet, in a stationary, and in a fluctuating noise condition, to cover the very different masker properties in typical listening conditions: quiet, low-dynamic maskers, high-dynamic masker. The main reason for using these “laboratory” signals and the SRT-50 instead of real noise recording and, e.g., the SRT-80, was the known high test-retest reliability of these tests. For individual predictions, the test-retest reliability of the employed speech recognition test sets the lower limit for the achievable prediction error in an evaluation with the data. That means, low measurement errors in the empirical data may facilitate or even enable falsification of the model predictions. An SRT of 0 dB at high noise levels in the test-specific noise condition, this is, with a stationary noise of identical long term spectrum than the speech signal, can be considered very problematic when normal-hearing listeners can achieve about -8 dB. If only half of the hearing loss in that condition in noise could be compensated (which would be a huge achievement), the measurable benefit would be only 4 dB. Considering that the benefit in SRT is calculated as the difference between two measurements, the targeted error of a single measurement should be less than 12⋅12⋅4dB≈1.41dB. Such low measurement errors, which can be achieved in SRT measurements with the matrix sentence test, would later enable to show individual benefits without averaging over groups of listeners, given the benefit was 4 dB. When adding to this consideration the need for a level-dependent evaluation, which is required to identify the class D loss according to Plomp (1978), one arrives at the test conditions which were already studied in Kollmeier et al. (2015) and that are depicted in Figure 2.\n\nThe second point, the selection of suitable listener profiles, is a bit more complex than it might initially appear. A sensible approach would be to take the individual profiles inferred from the psychoacoustic measurements by Schädler et al. (2020a) which are available online2 . The main problem with this approach is, that even with a small pure class A loss, the SRTs are generally not level-independent at high levels. This can already be observed for the fluctuating noise condition in the left panel of Figure 2. There, the simulations with a pure class A loss with hearing thresholds according to the standard profile N1 (corresponding to a very mild hearing loss) do not converge with the data of the normal-hearing profile None up to noise levels of 90 dB SPL. Hence, even for very small increases in hearing threshold, amplification improves the SRT in the fluctuating noise condition up to very high presentation levels. With the aim of clearly attributing compensation strategies to compensate a class A or a class D loss, this is highly undesirable. To clearly identify the compensation of a class D loss, simple linear amplification alone must not improve the SRT. The reason for the observed model behavior is that, for the Bisgaard profiles, the frequency range above the hearing threshold increases with the presentation level. While the limited frequency range can safely be assumed a factor contributing to a class D loss and has to be considered in a suitable listener profile, it must be avoided that the effectively used frequency range changes at high presentation levels. One option would be to low-pass filter the speech material. Another option is to define profiles with very steep sloping hearing loss functions. The former option would be very suitable to measure empirical data. The latter option is regarded cleaner from a modeling perspective, because it reduces the number of parameters that influence the SRT and results in a simpler and possibly better traceable model. Hence, listener profiles with normal hearing thresholds below, and infinite hearing loss above a given limit frequency are suitable for the considerations in this contribution. While the measurements from Schädler et al. (2020a) indicate that the level uncertainty might be frequency-dependent, profiles with frequency-dependent level uncertainty would add an additional dimension to a already complex matter. Also, the data in that study was from a non-representative group of 18 listeners of which it is unknown if extreme cases of class D loss were present. Hence, for the purpose of demonstrating the effectiveness of the compensation approach with PLATT and to discover its interactions and limits, idealized profiles that can be described by only two parameters, the frequency limit and (frequency-independent) level uncertainty, are preferable. In later measurements with human listeners, the limited frequency range can be achieved by low-pass filtering the signals to match the considered conditions.\n\nWith these considerations the following steps are enabled:\n\n• The applicable functional description of a process that is assumed to cause a class D hearing loss, the level uncertainty, can be used to design a matched functional compensation strategy.\n\n• Simulation experiments with FADE can be conducted which quantify the compensation of a class D loss as implemented with the level uncertainty as a function of frequency range and level uncertainty.\n\n• These experiments can later also be conducted with human listeners to test the hypothesis.\n\n\nMethods\n\nThe methods described in the following were used to simulate speech recognition experiments in stationary and fluctuating noise at different presentation levels for 16 listening profiles with class D hearing losses without and with the later proposed dynamic range expansion by PLATT including different degrees of expansion.\n\nThe speech material of the (male) German matrix sentence test (Wagener et al., 1999; Kollmeier et al., 2015) was used with two masker signals: the test-specific noise (called OLNOISE) and the fluctuating ICRA5-250 noise signal (Dreschler et al., 2001; Wagener et al., 2006). The matrix test, which exists in more than 20 languages, comprises 50 phonetically balanced common words of which sentences with a fixed syntax, such as “Peter got four large rings” or “Nina wants seven heavy tables”, are built. Typically, the SRT-50, this is the speech level that is required to correctly recognize 50% of the words, is measured with an adaptive procedure in experiments with human listeners. In this contribution, the speech and masker material was used to predicted the SRT-50 with FADE. The test-specific noise (OLNOISE) has the same long-term spectrum as the speech material. It can be assumed to mask the speech signal similarly well across all frequencies. At the SRT for normal hearing listeners, −7 dB (Hochmuth et al., 2015), this results in a noisy speech signal with a low dynamic range, where spectro-temporal maxima of the mixtures are dominated by the speech signal. The effect can be observed in Figure 3, where the log Mel-spectrogram of clean speech signal (upper panel) and the same speech signal with the OLNOISE masker at −7 dB SNR (center panel) are depicted. The ICRA5-250 noise is a speech-shaped noise which is co-modulated with speech-like temporal patterns in three independent frequency bands, where the pause duration was limited to 250 ms (Wagener et al., 2006). The empirical SRTs with this masker signal are usually more than 10 dB lower than in the corresponding test-specific noise condition (Hochmuth et al., 2015). At the SRT for listeners with normal hearing, −19 dB (Hochmuth et al., 2015), this results in a noisy speech signal with a high dynamic range, where the spectro-temporal maxima of the mixtures are dominated by the masker signal. This can be observed in the lower panel of Figure 3, where the log Mel-spectrogram of a speech signal with the ICRA5-250 masker at −19 dB SNR is depicted in the lower panel. To assess the presentation level dependency, both maskers are considered at presentation levels from 0 to 100 dB SPL in 10-dB steps. At 0 dB SPL presentation level, this corresponds to listening in quiet. The selected listening conditions reflect important dimensions of speech perception: listening in quiet, at low levels, and at high levels, as well as listening in stationary and fluctuating noise. All considered speech tests can also be performed human listeners.\n\nLog Mel-spectrograms of a clean German matrix sentence at 65 dB SPL (upper panel), of the same sentence in the stationary noise (center panel) and fluctuating noise (lower panel) conditions at the SNRs which correspond to the SRT listeners with normal hearing of speech, −7 and −19 dB, respectively.\n\nThe speech tests considered in the section Speech recognition tests were simulated with an ASR-based approach and their outcome, the SRT-50, was predicted based on the simulation results. The simulations were performed with the latest standard version of FADE3 , as described by Schädler et al. (2016a). In this contribution, FADE is used as a tool to predict the outcome of speech recognition tests, where the only change to the standard setup was the manipulation of the feature extraction stage which is explained in the section Listener profiles and the processing of the noisy speech signals with PLATT as explained in the section PLATT dynamic range manipulation. Hence, the FADE simulation method is only outlined here, and we refer the interested reader to the original description from Schädler et al. (2016a).\n\nPredictions with FADE are performed completely independently for each listening condition (masker, maskerlevel, hearing loss compensation, and hearing profile). There is no dependency on any empirically measured SRT, nor on predictions of the same model in other/reference conditions (and hence no need to define such). This means, with FADE, a single SRT of a speech recognition test for which no empirical data exists can be predicted. For the prediction of one outcome the following standard procedure as described by Schädler et al. (2016a) was used.\n\nAn ASR system was trained on a broad range of SNRs with noisy speech material form the considered condition, e.g. German matrix sentence test in OLNOISE for listener profile “P-8000-1” with no compensation. For this, a corpus of noisy speech material at different SNRs was generated from the clean matrix sentence test material and the masker signal, by adding randomly chosen masker signal fragments with the speech material. The noisy signals were processed with PLATT when an aided listening condition was considered. From the noisy (and optionally processed) speech signals, features were extracted, where this step included the implementation of the class D hearing loss, as described in the section Listener profiles. Subsequently, an ASR system using whole-word models implemented with Gaussian Mixture Models and Hidden Markov Models, was trained on the features. This resulted in 50 whole-word models for each training SNR. These models were then used with a language model that considers only valid matrix sentences (of which 105 exist) to recognize test sentences on a broad range of SNRs with noisy speech material from the same considered condition. For each combination of a training SNR and a test SNR, the transcriptions of the test sentences were evaluated in terms of the percentage of correctly recognized words. The resulting recognition result map (cf. left panel in Figure 7 in Schädler et al. (2016a) for an example), which contained the speech recognition performance of the ASR system depending on the training and testing SNRs in 3 dB steps, was queried for the SRT. For a given target recognition rate, e.g. 50% words correct, the lowest SNR at which this performance was achieved was interpolated from the data in the recognition result map and reported as the predicted SRT for the considered condition. The whole simulation process, including the creation of noisy speech material, an optional processing of this noisy speech material with PLATT, the feature extraction (which depends on the listener profile), the training of the ASR system, the recognition of the test sentences, and the evaluation of the recognition result map, was (independently) repeated for each considered condition.\n\nOutcome predictions of speech recognition tests as well as basic psychoacoustic tests with FADE were found to be close to the empirical results for listeners with normal hearing (Schädler et al., 2016a). As proposed by Kollmeier et al. (2016) and successfully used by Schädler et al. (2020a), impaired hearing was implemented in the ASR system by removing the information from the feature vectors that is presumably not available to listeners with impaired hearing. As discussed in the Introduction, two types of manipulations which induce class D hearing loss were considered: 1) a limitation of the frequency range, 2) an increase of level uncertainty. The effect of both parameters on the log Mel-spectrogram of a clean speech sample is depicted in Figure 4, where in the upper panel, the frequency range was limited to 8000 Hz and the level uncertainty was 1 dB. In the center panel, the level uncertainty was increased to 7 dB, compared to the upper panel. In the lower panel, the frequency range was additionally limited to 2000 Hz compared to the center panel. An amplification of the input signal increases all values in a log Mel-spectrograms by a constant value. Both manipulations introduce a level-independent loss of information and hence induce a class D loss. For the evaluation, upper frequency limits of 1000, 2000, 4000, and 8000 Hz were considered, where the class D loss decreases with high values. For the level uncertainty, frequency-independent values of 1, 7, 14, and 21 dB were considered, where the class D loss increases with high values. All combinations of both parameters result in 16 profiles from “P-8000-1” to “P-1000-21”. The former can be expected to be the best performing one, while the latter can be expected to be the worst performing one.\n\nThe first number in the profile encodes the upper frequency limit, in this example 8000 and 2000 Hz, and the second number indicates the level uncertainy, here 1 and 7 dB.\n\nIn this section, the patented (DE 10 2017 216 972 B4) PLATT dynamic range manipulation as it was conceived for a later implementation in a hearing device is described. The implementation was optimized to run in real-time on a Raspberry Pi 3 Model B to enable field studies with mobile hearing aid prototype hardware4 . The ability to expand spectral modulation frequencies in the range of 18 to 14cyclesERB is a feature that integrates naturally with the approach. Even if not strictly necessary for the goals of this contribution, the method is described here in detail to make statements about its ability to compensate a class D loss in the algorithmic context in which it might be later usable in a hearing device. To motivate the design decisions behind PLATT, which generally aims to preserve relevant speech modulations when compressing the dynamic range of a signal, this subsection comes with its own introductory part.\n\nIntroduction to the PLATT concept Conditions in which the available dynamic range for acoustic communication is reduced are rather the norm than the exception. For example, in a driving car, the lower limit of the available dynamic range is determined by the driving noise. Or in a library, the upper limit is given by the accepted sound levels in such an environment. And, importantly, the available dynamic range for communication is limited for listeners with impaired hearing. For a successful communication, it may be required to adapt a source signal, which may contain speech and non-speech parts, to the available dynamic range on the receiver side by dynamic range compression. But, in many real-time applications, the available temporal context to perform this operation is very limited.\n\nMulti-band dynamic range compressors used in hearing (aid) research (e.g. Grimm et al., 2015) statically map the input dynamic range to a reduced output dynamic range in a number of independent frequency bands. The compression is often applied with rather short attack time constants, e.g., 20 ms, with the aim to protect the user from high levels, while the release time constants are usually much longer, e.g. 100 ms to 1000 ms, with the aim to limit compression when it not desirable, i.e. during short speech pauses. However, no distinction is made whether the signal contains speech portions or not. Approaches which depend on a classification whether or not speech is present in the input signal, are prone to errors if the (speech-)signal-to-noise ratio (SNR) is low, that is, just when the classification result is most important. Approaches that require more than a few milliseconds of future temporal context cannot be used in applications which require low latency, such as, e.g., hearing devices. Regarding the speech intelligibility of processed signals, compression in a few wide frequency bands is preferred over compression in many narrow frequency bands, however, the recommended number of channels greatly varies (usually between 1 and 8) (Plomp, 1988; Dreschler, 1992; Hohmann and Kollmeier, 1995; Yund and Buckles, 1995; Moore et al., 1999; Souza, 2002). The fewer channels are used, the better the spectral dynamic, that is, the spectral contrast or spectral modulation, is preserved. Static dynamic range compression only preserves the spectral modulation within each independent frequency band, but not across bands, even if the dynamic range would be available. Also, fewer frequency channels reduce the need for sharp filters which would require long integration time constants and introduce additional latency.\n\nBustamante and Braida (1987) proposed to compress the first two principle components (PC1 and PC2) of the short-term speech spectrum, which were roughly representative of overall level and spectral tilt. With this approach, the frequency bands were not processed independently anymore, and the finer spectral structure was always preserved. Their analysis indicated that the highest intelligibility was obtained when audibility was improved and the relative spectral shapes of different speech sounds were preserved (Bustamante and Braida, 1987). In their concluding section, they recommended to investigate the enhancement of spectral differences while compressing level variations. Levitt and Neuman (1991) proposed an approach which decomposes and manipulates the short-term spectrum using a set of orthogonal polynomial functions with the aim to preserve important speech cues. Referring to the study of Bustamante and Braida (1987), Levitt and Neuman (1991) wrote: “Both studies showed that compression of the lowest order component (factor 1 in the principal-components method and the constant term in the orthogonal polynomial method, respectively) had by far the largest effect, and that compression of higher order components had little effect, if any.” The common idea behind these two studies was to linearly map and manipulate the spectral dimension of a suitable spectro-temporal representation with the aim of separating important from less important speech signal dynamic. However, both studies considered only clean speech signals, and hence did not consider the relevant portions of speech signal for their recognition in noise.\n\nThat the signal dynamic can be described as the difference of frequency-dependent short-term effective amplitudes, e.g., across time (temporal dynamic), across frequency (spectral dynamic), or both (spectro-temporal dynamic), raises the question which representation is most suitable to manipulate it. ASR systems are the technical solution to decode speech signals and hence provide a model for speech recognition. As outlined in the Introduction, the feature extraction stages of ASR systems provide representations of the speech dynamic that are well suited for the robust recognition of speech in noise. However, the often employed basis for the feature extraction stages, the log Mel-spectrogram, is not suited for low-latency signal processing due to its long integration window. The relatively long integration window of the log Mel-spectrogram serves two objectives: 1) obtain a sufficiently high frequency resolution to separate low-frequency signal content into approximately 1 ERB-wide bands, and 2) ensure that in voiced speech portions each signal frame contains at least one pulse (that is to remove the temporal fine-structure of the speech signal). Fortunately, these two aspects (sufficient spectral resolution for low frequencies and limited temporal resolution) are compatible and can be optimized for low-latency processing at the cost of a frequency-dependent group delay. In the following, the design of PLATT, a fast adaptive dynamic range manipulation scheme that takes the mentioned observations into account, is proposed, where the following three objectives were pursued:\n\n• Preservation and enhancement of spectral modulations which are assumed to be relevant for speech recognition\n\n• Low-latency and fast reaction time while minimizing audible artifacts\n\n• Adaptive limitation of the compression to the necessary minimum\n\nPLATT consists of three functional parts:\n\n1) An auditory-motivated frequency decomposition and re-synthesis of the audio signal, which allows to manipulate frequency- and time-dependent amplitudes in a perceptually relevant domain and helps to ensure that only limited audible artifacts can be introduced.\n\n2) Extraction of a spectro-temporal representation from the frequency-decomposed signal which is similar to those used for robust ASR and hence suitable for the analysis of the relevant spectral modulations.\n\n3) Adaptive calculation of frequency- and time-dependent gains from the spectro-temporal representation which uses compression only as required to provide high speech recognition performance when the available dynamic range on the output side is limited.\n\nFigure 5 illustrates the relations between the signal processing blocks that were used to implement this functionality. A detailed description is provided in the following. The exact implementation details are provided in a reference implementation that is written in C (cf. Data availability).\n\nDiagram illustrating the relations of the main signal processing blocks which were used to implement the signal analysis, manipulation, and re-synthesis with PLATT.\n\nFrequency decomposition & re-synthesis The frequency decomposition of the input signal is performed with a filter bank of fourth-order Gammatone filters. To get a set of frequencies which are relevant for (automatic) speech recognition, the center frequencies are chosen equidistantly on a Mel-frequency scale with half the distance that is commonly used for calculating MFCCs. Considering frequencies in the range from 64Hz to 16000Hz results in the following 78 +4 values: (64, 93,) 123, 155, 187, 221, 256, 293, 330, 370, 410, 453, 496, 542, 589, 638, 689, 742, 797, 854, 914, 975, 1039, 1105, 1174, 1245, 1319, 1396, 1476, 1559, 1645, 1734, 1827, 1923, 2023, 2127, 2235, 2346, 2462, 2583, 2708, 2838, 2972, 3112, 3257, 3408, 3565, 3727, 3896, 4071, 4253, 4441, 4637, 4840, 5051, 5270, 5498, 5734, 5979, 6233, 6497, 6771, 7056, 7352, 7658, 7977, 8307, 8650, 9006, 9376, 9760, 10158, 10572, 11001, 11447, 11909, 12390, 12888, 13406, 13943, (14501, 15080) Hz . Only the 78 filters with center frequencies from 123 Hz to 13943 Hz, spaced approximately 0.5 equivalent rectangular bandwidths (ERB), are used. The -10 dB-bandwidth of each fourth-order Gammatone filter is chosen to be equal to the difference of the frequencies two positions right and left to its center frequency, e.g., 221 Hz−93 Hz = 128 Hz for 155 Hz. The aim is to evenly cover the relevant frequency range with filters that have a bandwidth similar to auditory filters (≈ 1 ERB) and allow a trivial re-synthesis in the time domain by simple summation of all filter bank outputs. With this goal, the filter coefficients are determined as follows: The pole in the complex z-plane that describes the frequency-dependent properties of a first-order infinite impulse response Gammatone filter is calculated according to the formula\n\nReal part of the impulse responses of a subset of the normalized, phase-adjusted, fourth-order Gammatone filters that were employed for the frequency decomposition, and the (scaled) sum of the impulse responses of all employed Gammatone filters.\n\nAbsolute values of the transfer functions corresponding to the impulse responses shown in Figure 6 and the absolute value of the joint transfer function of all filters (including those not shown).\n\nSpectro-temporal signal representation A representation of the spectral dynamic which encodes information similar to a log Mel-spectrogram is determined as follows, based on the real-valued output of the filter bank. For each filter output, the values are held for 15 ms and decay subsequently with a rate of 1dBms, if the held value is above the current value. This approach approximately extracts the temporal envelope of each channel while preserving fast increases in amplitude (on-sets). Hence, the exact timing (or temporal fine structure) is removed from this representation, and only the local maximum values remain as an estimate of the maximum amplitude (or displacement) of an oscillatory system with properties similar to those of the employed Gammatone filters. This representation can be down-sampled by any factor which reduces the sample rate to 115ms≈67Hz or higher, without missing any local maximum value. The encoded information is very similar to the information encoded in features for ASR, where updated spectral values are determined every 10ms in frequency-bands which are equally spaced on a Mel-scale. Pilot experiments confirm that the proposed representation (unpublished data), down-sampled to 100Hz, achieves very similar simulation results in a range of speech recognition experiments with FADE. The use in a hearing device, however, requires faster updates which is why the representation is down-sampled to 1000Hz, that is, an update of the 78 spectral values is calculated every 1ms. In Figure 8, the spectro-temporal representation used in PLATT and the log Mel-spectrogram of a clean speech sample at 65 dB SPL are shown. Compared to the log Mel-spectrogram, the proposed spectro-temporal representation has a 10-times higher temporal resolution (visible at the on-sets), where, however, the temporal fine structure is effectively removed. The off-sets are not as prominent because the maximum tracking only allows a decrease by 1dBms=100 dB100 ms. Also, the proposed spectro-temporal representation has approximately twice the number of frequency channels, 78 compared to 36 with the the log Mel-spectrogram. The aim of the spectral oversampling is to provide headroom for spectral modulation manipulations.\n\nComparison of spectro-temporal representations: In the upper panel a log Mel-spectrogram as it is used in FADE of a clean speech sample at 65dB SPL, and in the lower panel the presented spectro-temporal representation that is used in PLATT of the same sentence.\n\nBecause a pure tone is not changed in amplitude by a filter of the employed Gammatone filter bank when it matches the filters center frequency, the calibration of its input and output values is identical. Assuming a calibrated setup, only values above the frequency-dependent normal hearing threshold, as defined by the International Organization for Standardization in its standard 226:2003 (ISO 226, 2003), are considered in the representation; values below the normal hearing threshold are replaced by the corresponding threshold value. This effectively removes spectro-temporal modulations that cannot be perceived by listeners with normal hearing from the proposed spectro-temporal signal representation. The effect can be observed in the high frequency range ( >8000 Hz) in Figure 8, where the speech signal has no energy.\n\nAdaptive spectral gain The adaptive determination of time- and frequency-dependent gains takes into account the current spectral input dynamic and the currently available output dynamic. It aims to minimize the compression with the constraint to avoid masking the signal parts which could carry important (speech) information. It also allows to expand the spectral modulations that are assumed to be important for speech recognition and trade the such increased signal dynamic against an increased compression of less relevant signal dynamic.\n\nThe spectral input dynamic is analyzed with spectral modulation low-pass filters. For this, each vector of the 78 spectral values is convolved with Hanning windows of the following widths to obtain increasingly spectrally smoothed versions of the initial vector: 8, 16, 32, and 64, which approximately correspond to a full width at half maximum (FWHM) of 2, 4, 8, and 16 ERB, respectively. The left panel of Figure 9 shows an example of the spectral analysis for a signal which consists of two pure tones, of 500 Hz and 2000 Hz. The input spectral values are depicted in black, the smoothed versions with ascending widths of the Hanning window in increasingly lighter shades of gray. The differences between the curves of increasingly smoothed spectral representations are indicated with the digits 1 to 4. Because the difference of two low-pass filters is a band-pass filter, the differences 1, 2, 3, and 4 can be interpreted as the result of a spectral modulation band-pass filtering which roughly contains the respective spectral modulation frequencies 1) above 14, 2) from 14 to 18, 3) from 18 to 116, and 4) from 116 to 132cyclesERB, respectively. By definition, the original vector of spectral values (cf. black line in left panel of Figure 9) can be recovered by adding the differences 1 to 4 to the low-pass filtered spectral values which contain the spectral modulation frequencies below 116cyclesERB (cf. lightest line in left panel of Figure 9). In the following, the vector of low-pass filtered spectral values which contains the lowest modulation frequencies is referred to as the base layer. The base layer contains a very coarse description of the spectral dynamic and resolves spectral patterns larger than approximately 16 ERB. The other layers, the differences 4 to 1, resolve the spectral dynamic which is not described in the base layer with decreasing pattern sizes.\n\nRight Panel: Gains required to map the input dynamic to the reconstruction stages of the output dynamic (final gains are black). Numbers indicate the differences.\n\nThe base layer needs to be mapped from the input dynamic range to the output dynamic range. For the application in a hearing device, the mapping of the base layer could be performed with a prescription rule. In addition, the limits of the input and output dynamic ranges need to be defined. Here, for the input, a dynamic range that is probably relevant for normal hearing listeners is assumed: A frequency-independent uncomfortable level of 105dB SPL as the upper limit, and levels of 25dB SPL from 500Hz to 4kHz, and 30dB above 8kHz and below 250Hz, as the lower limit. The assumed input dynamic range is indicated with triangles in the left panel in Figure 9. For the output, an exemplary reduced dynamic range is assumed, which is arbitrarily limited to frequency-independent levels from 50 to 90dB SPL; resembling elevated hearing thresholds due to environmental noise or impaired hearing and a lower acceptance of high levels. The targeted output dynamic range is indicated with triangles in the center panel in Figure 9. The mapping of the base layer can be independent from the input and output dynamic range definitions. In the context of a hearing device, the mapping of the base layer will mainly determine the output levels and strongly affect loudness perception, while the lower limit of the output dynamic range (to be chosen related to the individual hearing thresholds) will strongly affect speech recognition performance. The defined output dynamic range is the reservoir that can be used by PLATT to map the input dynamic.\n\nIn our example in Figure 9, let’s assume the base layer was mapped linearly from the defined input dynamic range to the defined output dynamic range. The base layer is depicted as the lightest gray line in the left panel, and the mapped base layer as the lightest gray line in the center panel. The gains which would be theoretically required to achieve such a smooth output dynamic, given the black line in the left panel as the input dynamic, are depicted as the lightest gray line in the right panel. But such an extreme compression of the spectral dynamic would introduce audible artifacts and would most likely have a negative effect on speech recognition performance. Hence, the more of the remaining original signal dynamic described by the differences 1 to 4 is added back to the mapped base layer, the less compression will be needed, and the better spectral modulation patterns will be preserved. However, unconditionally adding the whole dynamic that is encoded in the differences 1 to 4 could result in output levels below the lower limit of the output dynamic range, which might not contribute to speech recognition anymore, or above the upper limit of the output dynamic range, which might lead to undesirably high output levels. A good compromise in the fundamental conflict that too much and too little compression can both result in sub-optimal speech recognition performance requires a compression management which depends on the current spectral input dynamic and the currently available output dynamic.\n\nTo prefer spectral patterns which are important for robust (automatic) speech recognition, the differences corresponding to high spectral modulation frequencies are added first. The highest spectral modulation frequencies are encoded in difference 1 and account only for a very small part of the total dynamic, which is why it is added unconditionally. The example with two pure tones is an extreme one which assesses the maximum dynamic that can be encoded in each difference, which is about 6 dB for difference 1. The corresponding output dynamic, when only adding difference 1 to the base layer, can be observed in the center panel of Figure 9 as the light gray curve which only deviates slightly from the base layer. Probably the most important spectral modulation frequencies describe spectral patterns between 2 and 4 ERB and are mainly encoded in difference 2, which is why it is also added unconditionally. To protect this difference, which encodes a maximum dynamic of less than 9 dB, against a hearing loss of class D as implemented in FADE with the level uncertainty, it can be expanded by a factor greater than 1 prior to adding it to the base layer. The expansion of the difference 2 could increase the total output signal dynamic, which however can often be compensated by an increased compression of the remaining differences 3 and 4, which encode larger part of the signal dynamic, compared to difference 2.\n\nThe remaining differences 3, and subsequently 4, are added conditionally and possibly partially according to the following constraints: 1) For each frequency, the respective difference is multiplied with the highest possible factor between 0 and 1 for which its addition to the current output dynamic (which already includes the base layer and differences 1 and 2) does not result in output values above the upper limit or below the lower limit. For example, if the current value is 80dB SPL, the difference +30dB SPL, and the limit 90dB SPL, only a third of difference can be added and the factor is 13. This rule is not sufficient because, when applied independently for each frequency band, it would result in a total loss of high spectral modulation frequencies in the frequency regions where no output dynamic range is left to add the difference; the already added spectral modulations needs to be protected from being compressed. Hence, 2) the minimum factor (highest compression) is propagated along the frequency axis by adding values of normalized Hanning windows of FWHM of 6 and 12 samples for difference 3 and 4, respectively. For example, if the factor according to 1) turns out to be zero at the center frequency of 2708Hz when adding difference 3, the factor at 2346Hz and 3112Hz can be at most 0.5 and below 2023Hz and above 3565Hz again 1. The propagation ensures that neighboring channels are compressed similarly and protects any higher spectral modulation frequencies. The final desired spectral output levels are described by the sum of the mapped base layer, the unconditionally added differences 1 and 2, and the conditionally compressed differences 3 and 4. The frequency-dependent gain needed to achieve the desired spectral output levels is the difference of the spectral output levels (black curve in the center panel in Figure 9) and spectral input levels (black curve in the left panel in Figure 9). The final frequency-dependent gain is plotted in black in the right panel of Figure 9 along with the partial gains that would theoretically be needed after the cumulative addition of only differences 1 to 3 to the base layer in increasingly darker gray shades. The final frequency-dependent gain is then applied to the output of the Gammatone filter bank with the limitation of the rate of change to 24 dB per period of the corresponding center-frequencies.\n\nAdmittedly, in our example with the two pure tones, there is only energy at 500 Hz and 2000 Hz, and hence only the level of the two tones will be changed, while the gains at other frequencies will have no effect. However, this signal creates a pattern of extreme spectral modulation in the proposed signal analysis, and hence is well suited to illustrate how PLATT works. For a signal with only low spectral modulations, no conditional compression would be required.\n\nSummary of PLATT dynamic range manipulation With PLATT, compression is only applied if the available output dynamic range is less than required to represent the input signal dynamic. The main effect can be observed in Figure 10, where the calculated gains for high (solid curves) and reduced (dotted curves) spectral input dynamic are shown for an exemplarily reduced output dynamic range, as indicated by the triangles. While the signals with high spectral dynamic are compressed (observed here as different gains for different frequencies) to make relevant signal portions audible and avoid excessively high output levels, the signal with low spectral dynamic is not (observed here as similar gains for different frequencies), because it fits in the available output dynamic range without compression. Only low spectral modulation frequencies, which are less important for speech recognition, are conditionally compressed, while the important higher spectral modulation frequencies can be even expanded. An efficient implementation of spectral modulation filtering in the time-domain is possible with reasonably low latency, fast reaction times, and probably little audible artifacts due to the auditory-motivated signal decomposition and re-synthesis.\n\nLeft panel: Spectral input dynamic with two pure tones at different presentation levels (gray shaded solid curves), and with white noise added (dotted curve). Center panel: Corresponding spectral output levels. Right panel: Corresponding gains.\n\nCompensation of level uncertainty with PLATT The possibility to selectively expand the spectral modulation patterns between 2 and 4 ERB with PLATT was used to protect these patterns against the effect of the level uncertainty as implemented in FADE. With the aim to best decouple the evaluation of the expansion from the conditional compression feature of PLATT, the available output dynamic range was set to match the input dynamic range, and the base layer mapping was set to identity. This minimizes compression and effectively disables the fine-grained compression management for all but very low and very high signal levels. Nonetheless, in view of expanding a part of the signal dynamic, such a management will probably be needed in an application with a limited output dynamic range to counter the expansion with a possible compression of other parts of the signal dynamic.\n\nExpansion factors of 2, 4, 6, and 8 were considered for the evaluation, and the corresponding compensation conditions are referred to as PLATT-2 to PLATT-8 in the remainder of the manuscript. The effect of processing noisy speech signals at 0 dB SNR with PLATT-6 is illustrated in Figure 11. In the top row, log Mel-spectrograms of a speech signal in the stationary and fluctuating noise at 0 dB SNR are depicted in the left and right panel, respectively. In the center row, the same log Mel-spectrograms are shown, but with a level uncertainty of 7 dB. In the bottom row, log Mel-spectrograms of the same signals, but processed with PLATT-6 are shown with a level uncertainty of 7 dB. Especially for the stationary noise, some of the speech patterns are better distinguishable in the log Mel-spectrogram with the added level uncertainty after the processing. This justifies the expectation that the expansion might protect important speech patterns against level uncertainty. With the fluctuating masker, the speech patterns are not as easily distinguishable from the background noise. But comparing the representation of the unprocessed (center) and processed (bottom) signals, more of the patterns that are hidden by the level uncertainty in the unprocessed signal are discernible in the processed variant. This illustrates that the expansion does not selectively process the speech portions of the signal but generally protects the spectral modulation patterns between 2 and 4 ERB against the level uncertainty, independently of what caused these patterns. Also observable, the processing with PLATT-6 results in a possible increase in the output level. This affirms the need to evaluate the method in a context where simple time-invariant linear amplification will not change the speech recognition performance and thus decouple the effect of the expansion from the effect of any linear amplification.\n\nThe just described effects of an expansion of the spectral modulation patterns between 2 and 4 ERB on noisy speech signals were quantified in simulation experiments with FADE. For this, SRTs with the German matrix sentence test were simulated for all combinations of considered noise maskers (OLNOISE, ICRA5-250), noise presentation levels (0, 10, 20, ..., 100 dB SPL), listener profiles (P-8000-1 through P-1000-21), and compensations (none, and PLATT-2 through PLATT-8); summing a total of 1760 outcome predictions with FADE.\n\nIllustration of the effect of the dynamic range manipulation with PLATT-6: Log Mel-spectrograms of noisy speech in stationary (left column) and fluctuating noise (right column) at 0 dB SNR (top row), the same log Mel-spectrograms with level uncertainty of 7 dB (center row), and log Mel-spectrograms with level uncertainty of 7 dB of the same signals processed with PLATT-6, that is with an expansion factor of 6.\n\nKey simulation outcomes are presented as “Plomp curves”, that is, SRT-50s in dB SPL as a function of the noise presentation level, analog to Figure 2. On the one hand, this depiction allows to assess the effect of the (noise) presentation level on the predicted SRTs. On the other hand, it also allows to quantify improvements in SRT in aided conditions over a given reference condition, e.g., normal hearing or an unaided condition. Because not all 1760 data points can presented in graphical form in this contribution, a summary of the achieved improvements in SRT at high presentation levels, at which we can confidently assume that linear time-invariant amplification cannot improve the SRT, are presented in the form of a table for all listener profiles and compensations. For key listener profiles, psychometric functions were obtained by simulating SRT-20 to SRT-90 to assess the SNR-dependency of PLATT. The main interest here was if the effect of the PLATT compensation is different for higher SRTs, which are more realistic for conversations.\n\n\nResults\n\nTwo modifications were used to implement a hearing loss of class D, the limitation of the frequency range up to a limit frequency, and the increase of the level uncertainty. Their separate effect on simulated SRTs is shown in Figure 12 and Figure 13, respectively.\n\nThe dotted lines indicate an SNR of 0 dB. The corresponding level-dependent differences in SRT compared the profile plotted in black (here profile P-8000-1) are depicted in panels two and four.\n\nAnalog to Figure 12.\n\nThe limitation of the available frequency range affected the simulated SRTs in the fluctuating noise condition much more than in the stationary noise condition when assuming a level uncertainty of 1 dB (cf. Figure 12). In the stationary noise condition, the limitation of the frequency range to 4000 Hz did not have an effect on the simulated outcome of the German matrix sentence test. A reduction to 2000 Hz resulted in a small, mostly level-independent increase in SRT of about 1 dB, and a further reduction of the frequency range to 1000 Hz, resulted in a further increase of about 1 dB. Hence, limiting the frequency range while assuming a level uncertainty of 1 dB had relatively little effect on the simulated SRTs. A different picture can be observed in the fluctuating noise condition. There, the reduction of the frequency range had a large, and level-dependent effect on the simulated SRTs. The effect of the reduction increases with the higher presentation level and stabilizes at levels above 60 dB SPL. At these levels, the increase in SRT was about 3, 6, and 15 dB for a limitation to 4000, 2000, 1000 Hz, respectively.\n\nThe increase of the level uncertainty also affected the simulated SRTs in the fluctuating noise conditions more than in the stationary noise condition (cf. Figure 13). The effect was not as level-independent at low presentation levels as one might have expect. This indicates an interaction between the implementation of the absolute hearing threshold and the level uncertainty of about 5 dB. The behavior could already be observed in the data from Kollmeier et al. (2016) (cf. right panel in Figure 2). However, in this contribution, the focus does not lie on the interactions of the level uncertainty with the absolute hearing threshold but effective elimination of the absolute hearing threshold as a factor from the evaluation. In the fluctuating noise condition, this is the case for presentation levels of 70 dB SPL and above, where the differences (cf. panel four in Figure 13) stabilize. At these levels, the increase in SRT was about 4, 9, and 12 dB for level uncertainties of 7, 14, and 21 dB, respectively. In the stationary noise condition, the corresponding increases were lower, with about 1, 4, and 6 dB for level uncertainties of 7, 14, and 21 dB, respectively.\n\nNeither implementation alone achieves to increase the SRT to above 0 dB with the considered parameter values. In listeners with impaired hearing, we assume that both factors, the level uncertainty as well as the limited frequency range, contribute to the class D loss. All combinations of both parameters were considered, of which a few are presented in Figure 14 and 15 to assess their interaction. In Figure 14, the simulation results for increased values of level uncertainty are shown when the frequency range was limited to 1000 Hz. Comparing the results to Figure 13 (limitation to 8000 Hz), the limitation of the frequency range increased the effect of the level uncertainty in the stationary noise condition, while the effect in the fluctuating noise condition remained similar. In the stationary noise condition, the average increase in SRT at levels above 70 dB was about 3, 7, and 11 dB for level uncertainties of 7, 14, and 21 dB, respectively. In the fluctuating noise condition, the corresponding increases were very similar, with 4, 7, and 11 dB for level uncertainties of 7, 14, and 21 dB, respectively.\n\nAnalog to Figure 12.\n\nAnalog to Figure 12.\n\nIt cannot be expected that a partial expansion of the signal dynamic (e.g. with PLATT) will compensate the increase in SRT due to limiting the frequency range. However, the simulations suggest an interaction of the level uncertainty with the limited frequency range, more specifically, that an increase in level uncertainty would be worse in the stationary noise condition if the frequency range is limited. This is due to the strongly non-linear nature of the (automatic) speech recognition process, which can be interpreted to involve a forward error correction scheme that integrates over frequency and over time, and which needs to fail in order to achieve an error rate as high as 50%. Forward error correction requires redundancy, which is reduced by limiting the frequency range. The simulation result shows that considering level uncertainty and a limitation of the frequency range separately would not show the full picture.\n\nTo graphically present effect of the PLATT compensation on the simulated SRTs, the mixed profiles P-4000-7, P-2000-14, and P-1000-21 were used. This choice reflects the assumption that the limitation of the frequency range (due to the audiogram) and an increase in level uncertainty are probably correlated to some extent. The simulation results with these listener profiles without PLATT compensation are shown in Figure 15. Compared to the normal-hearing configuration, in the stationary noise condition, the average increases in SRT at levels above 70 dB were about 2, 6, and 13 dB with the profiles P-4000-7, P-2000-14, and P-1000-21, respectively. In the fluctuating noise condition, the corresponding increases were much more pronounced, with 7, 14, and 26 dB with the profiles P-4000-7, P-2000-14, and P-1000-21, respectively.\n\nThe effect of the expansion with PLATT on the simulation results for listener profiles P-4000-7, P-2000-14, and P-1000-21 is presented in Figures 16, 17, and 18, respectively. The blue, red, and yellow lines indicate the simulated speech recognition performance with compensations PLATT-2, PLATT-4, and PLATT-6, respectively. The black lines indicate the corresponding unaided reference conditions, while the purple lines indicate the simulated performance of profile P-8000-1, that is, the performance of a listener with normal hearing.\n\nThe dotted lines indicate an SNR of 0 dB. The corresponding level-dependent differences in SRT compared the profile plotted in black (here the unaided profile P-4000-7) are depicted in panels two and four. The data with profile P-8000-1 (normal hearing) was added as an orientation for normal-hearing performance.\n\nAnalog to Figure 16.\n\nAnalog to Figure 16.\n\nThe expansion with PLATT improved the SRTs in almost all simulated conditions. The benefit in SRT due to PLATT in was generally higher in the fluctuating noise condition than in the stationary noise condition. Higher expansion factors strongly tended to result in higher benefits, however, not in all conditions. As expected, the observed compensations were partial, that is, the simulated performance of the profile with normal hearing were not restored. For a quantitative analysis, the results are interpreted only at the levels at which an improvement in SRT due to simple linear amplification can be ruled out.\n\nThe curves stabilize at high SRTs, that is, a further increase in levels does not improve the SRT anymore. In the stationary noise condition, this is the case for presentation levels of 40 dB SPL and above. But even above this level, the curves show some variability, which is due to the stochastic nature of the simulation process. The plotted data points were derived as the difference of two independent simulations. Assuming a random (normally distributed) prediction error of 0.5 dB, this would lead to a random error of the plotted data points of approximately ±0.71 dB, that is for 5 of 100 simulation results, on average, the error will be larger than ±1.96 ⋅ 0.71 ≈ ±1.39 dB. Hence, “stabilized” refers to reaching a state which may allow to assume that the remaining variability is due to the random error. In the fluctuating noise condition, the random error is increased, because of the generally shallower slope of the psychometric function in this condition. There, the curve of the normal-hearing profile stabilizes at levels of about 70 dB. The curves of the unaided and compensated simulations already stabilize at lower levels. Hence, at noise presentation levels of 70, 80, and 90 dB SPL an improvement in SRT due to simple linear amplification can be ruled out.\n\nFor a break-down of the simulation results, the average improvements at noise presentation levels of 70, 80, and 90 dB SPL were calculated and are reported in Table 1.\n\nNH indicates the normal-hearing listener profile.\n\nThe tables present the simulated benefits in SRT using PLATT with expansion factors 2, 4, 6, and 8, for all 16 considered listener profiles, sorted by the limit frequency. In the last column, the benefit in SRT that corresponds to the normal-hearing (NH) profile is presented as an orientation to assess the proportion of the total class D loss that be compensated. Positive values indicate an improvement, that is, a lower SRT. An orientative value for the random error of the presented simulation data in Table 1 is 1 dB.\n\nFor listener profiles with a level uncertainty of 1 dB (P--1), only small benefits were observed. In the stationary noise condition, processing the signals with PLATT had no effect on the SRTs (≤0.4 dB). In the fluctuating noise condition, small positive and negative benefits (min. −2 dB and max. 2 dB) were observed; the SRTs for the listener profile P-1000-1 improved (lower SRTs) and the SRTs for the listener profiles P-8000-1 and P-4000-1 increased (negative improvement). This effect was most pronounced with PLATT-8 and PLATT-6, less pronounced with PLATT-4, and could not be observed with PLATT-2, that is, it depended on the expansion factor. The processing with PLATT, by increasing the amplitude of certain spectral modulations, decreases the prominence of (or “masks”) other modulation patterns, and it introduces a possible leak of information across frequencies, e.g., from >1000 Hz to <1000 Hz. While the masking of other modulation patterns could explain the detrimental effect of PLATT for the profiles P-8000-1 and P-4000-1, the leakage of information from higher frequencies could explain the improvement for profile P-1000-1. Despite these small variations of the SRTs due to the processing with PLATT in the fluctuating noise condition, one might feel comfortable to state: As expected, the effect of a limited frequency range (an increase in SRT that cannot be compensated by simple amplification) cannot be compensated with the expansion performed by PLATT.\n\nThe data in the column NH, that is, the difference in SRT between the simulation with a specific listener profile and the normal-hearing profile, shows again how different the simulation results are in the stationary and fluctuating noise condition. While limiting the frequency range to 1000 Hz (P-1000-1) only resulted in a surprisingly small increase in SRT, namely by 2.3 dB, the same modification increased the SRT in the fluctuating noise condition by 15.0 dB. This reaffirms the use of different noise maskers to assess speech in noise recognition performance.\n\nInterpreting the addition of maskers to a speech signal as a frequency-dependent removal of information, the results indicate that the information in the mid-frequency range (1000-4000 Hz), was much more relevant in the considered fluctuating noise condition (ICRA5-250) than in the stationary noise condition (OLNOISE). In other words, in the stationary noise condition, this mid-frequency portion was mostly redundant at the SRT, and the ASR system could discriminate the 50 words of the matrix sentence test almost equally well only using the low-frequency information. This was not the case for the fluctuating noise condition, where the mid-frequency portions contributed a substantial part of the information to achieve low SRTs (say, less than -15 dB). For the following presentation of the benefits with PLATT, it is important to keep in mind that this missing information, by design, cannot be compensated with PLATT. That means, when considering the maximum achievable improvement for a specific profile, the increase in SRT due to limiting the frequency range was disregarded. For example, with profile P-2000-14 in the fluctuating noise condition, the difference to the normal-hearing SRT is 14.3 dB; improving the SRT by 14.3 dB the performance of the normal-hearing profile would be achieved. When only considering the increase in SRT due to limiting the frequency range, that is profile P-2000-1, the difference to the normal-hearing SRT is 6.5 dB. Hence, the maximum achievable improvement for profile P-2000-14 is then estimated by the difference 14.3−6.5 = 7.8 dB.\n\nFor profiles with increased level uncertainty (P--7, P--14, and P--21), the average benefits in SRT due to using PLATT were positive and ranged from 0.5 to 7.8 dB. For these profiles, the lowest improvements were found with PLATT-2, and the highest improvements often, but not always, with PLATT-8. Overall, in both noise conditions, a very strong relation between the increase in level uncertainty and the improvement in SRT due to using PLATT could be observed. Combined, there was strong general tendency that with higher level uncertainty and with higher expansion factors larger improvements were observed. Exceptions were observed only for the profiles with a level uncertainty of 7 dB (P-*-7), where higher expansion factors did not further increase the improvement. This data supports the sensible expectation that lower expansion factors are sufficient (or even optimal) to compensate lower values of level uncertainty. For profiles with a level uncertainty of 14 or 21 dB, the improvements always increased together with the expansion factor. With high values of level uncertainty, higher expansion factors might further improve the SRT; however, increasing the dynamic of a signal portion eight fold might have undesirable collateral effects which are discussed later.\n\nIn absolute terms, the improvements were generally larger in the fluctuating noise condition than in the stationary noise condition. For example with the extreme profile P-1000-21 and PLATT-8 compensation, the improvement was 5.3 dB in the stationary noise condition, and 7.8 dB in the fluctuating one. However, relating the improvement to the performance with the normal-hearing profile (rightmost column in Table 1), 5.3 dB of a total class D loss of 13.1 dB were compensated in the stationary noise condition and “only” 7.8 dB of a total class D loss of 26.3 dB. While this interpretation correctly reflects the proportion of the total class D loss that was compensated, it does not reflect that a part of the class D loss cannot be compensated by expansion approach with PLATT by design. As explained earlier, the portion of the class D loss due to limiting the frequency range to 1000 Hz is very different in both maskers. To evaluate the achieved improvements with respect to the maximum achievable improvement, the class D loss due to limiting the frequency range needs to be disregarded. In the context of the maximum achievable improvement, PLATT-8 compensated 5.3 dB of (13.1−2.3 =)10.8 dB and 7.8 dB of (26.3−15.0 =)11.3 dB of the class D loss due to a level uncertainty of 21 dB in the stationary and fluctuating noise condition, respectively. For the intermediate mixed profile P-2000-14, PLATT-6 compensated 3.0 dB of (6.5−1.2 =)5.3 dB and 5.3 dB of (14.3−6.5 =)7.8 dB in the stationary and fluctuating noise condition, respectively. And for the least extreme mixed profile P-4000-7, PLATT-4 compensated 1.0 dB of (1.5−−0.1 =)1.6 dB, and 2.7 dB of (7.2−3.2 =)4.0 dB. Hence, in relative terms, over a broad range of assumed parameters, PLATT expansion compensated at least about half of the class D loss caused by an increase of a level uncertainty.\n\nThe absolute improvements due to PLATT tended to increase with an increased limitation of the frequency range. The effect of the level uncertainty increased with an increasing limitation of the frequency range. Based on the presented data, however, it is difficult to make statements about the frequency-dependency of an optimal expansion factor because of the diverse non-linear interactions between the considered parameters.\n\nWe (humans) generally prefer to have conversations at higher SNRs than the SRT-50, that is, at SNRs at which more than 50% of the words can be correctly recognized; which might correspond to something like the SRT-80. The SRT-80 can be simulated, but it cannot be as efficiently and accurately measured in listening experiments as the SRT-50, because of the shallower slope of the psychometric function at the SRT-80. The main reason for simulating the SRT-50 was that it can be accurately measured in later listening experiments with human listeners, and accurate measurements are a requirement to show effects as small as 1 dB. To assess if the improvements would (at least according to the model) translate to improvements at SRTs preferred in real conversations, the psychometric functions of aided and unaided conditions were compared. Segments of psychometric functions were obtained by evaluating simulations at SRT-20, SRT-25, ..., SRT-90. Figures 19, 20, and 21 present the unaided and aided psychometric functions for the mixed profiles P-4000-7, P-2000-14, and P-1000-21, respectively. The simulation results in the unaided conditions are plotted in black, the corresponding simulation results with PLATT2, PLATT4, PLATT6, and PLATT8 expansion in blue, red, yellow, and purple color, respectively. As expected, the slopes in the fluctuating noise conditions (right panels) were shallower than the slopes in the stationary noise condition (left panel). Also, as expected, the data points in the fluctuating noise conditions were more noisy, due to the greater variability in the spectro-temporal distribution of the masker energy. This variability could be decreased by increasing the amount of training data and testing data. Within the uncertainty due to this variability, no reduction in improvement between SRT-50 and SRT-80 can be observed for listener profile P-4000-7. For profile P-2000-14 (in Figure 20), where the improvements are larger, there was a trend towards a slight increase in slope with higher expansion factors. This indicates, that the improvements due to the expansion with PLATT of the SRT-80 might be slightly larger than the corresponding improvement of the SRT-50. This trend was confirmed by the data with the profile P-1000-21 in Figure 21. According to the these simulations, the expansion with PLATT was found to improve the simulated SRT-80 to the same extent or even more than the SRT-50.\n\nThe dotted and dashed lines indicate a word recognition rate of 50% and 80% correct, respectively.\n\nAnalog to Figure 19.\n\nAnalog to Figure 19.\n\n\nDiscussion\n\nThe presented experimental results were derived using a model of auditory perception, more specifically, a model of impaired human speech recognition based on automatic speech recognition. The modeling approach with FADE brings assumptions about the impaired human speech recognition process into a form in which they can be tested by comparing predictions with empirical data. The employed model, the framework for auditory discrimination experiments (FADE) such as it was used by Schädler et al. (2020a), was already evaluated with respect to predictions of the individual aided speech recognition performance of listeners with impaired hearing. There, as elaborated in the Introduction, an important assumption was that the part of the hearing loss which cannot be explained by the absolute hearing threshold, that is, the missing piece to describe the effect of hearing loss on speech recognition in noise, can be explained by the level uncertainty. Another assumption was, that this model parameter also affects tone in noise perception and hence its value could be inferred from tone in noise detection tests. While the results supported this hypothesis, evidence was not sufficient to rule out other mechanisms that would also increase the SRTs in noise. This is a fundamental problem in modeling the individual speech recognition performance. While the quantity that is predicted by the model, the SRT, can be measured in experiments with human listeners, the outcome of such measurements still depends on many correlated and non-linearly interacting parameters; some of which cannot be controlled very well, such as, e.g., attention. And even if the experimental results were measured with the most accurate methods, the measurement errors include this uncontrollable (human) variability which can increase the required amount of data to falsify hypotheses to infeasible regions. This is especially true for hypotheses which predict relatively small effects. Hence, there is reasonable doubt about whether the removal of information in listeners with impaired hearing is really well described by the level uncertainty, or if it just coincidentally increased the SRT in the correct conditions.\n\nTo more specifically test if the level uncertainty is suitable to describe the effect of hearing loss on speech recognition in noise, a promising approach is to interact with it. The expansion of PLATT was specifically designed to interact with—namely compensate—the effect of the level uncertainty. The modeled data clearly showed this interaction. If this specific interaction was found in empirical data, it would be strongly supportive for the hypothesis of the existence of a mechanism in the human auditory system similar to the level uncertainty. Beyond the academical interest in the suitability of the assumptions to describe impaired human speech recognition performance, a positive result would have immediate practical implications for the design of hearing loss compensation strategies.\n\nLet’s remember that the goal was not to test if the expansion with PLATT improves speech recognition performance of listeners with impaired hearing. For that, measurements with listeners with impaired hearing will be necessary. The aim of this contribution was to:\n\nA) Present an approach which is able to partially compensate a class D loss as implemented with the level uncertainty in FADE, and\n\nB) objectively evaluate this approach and come up with testable quantitative hypothesis on the benefit in noisy listening conditions.\n\nThe latter aim, in other words, was to guide the planning of the measurements with listeners with impaired hearing towards optimal evidence. Hence, the following discussion is oriented towards the planning of a suitable experiment.\n\nPlomp (1978) did not distinguish mechanisms causing the postulated class D hearing loss which he used to describe hearing loss in noise. His D component described the total loss in SRT in noise due to impaired hearing, also referred to as speech hearing loss D (SHLD). He reported average empirical values of SHLD from five investigations which range from as low as 1 dB to above 10 dB (cf. TABLE III in Plomp (1978)). In stationary noises (average speech spectrum noise, white noise, and airplane cockpit noise) the maximum average values for SHLD were 6 to 8 dB. In fluctuating noises (interfering talker and voice babble) the maximum average values were larger, up to 14 dB. The increase in SRT in noise was related to the increase in SRT in quiet SHLA+D in Figure 8 in Plomp (1978), which showed that every 3-dB increase of SHLA+D comes with a 1-dB increase of SHLD. This data indicated that, on average, about a third of the hearing loss in quiet (SHLA+D) was incompensable by simple amplification; that would be a lot. Listeners with speech hearing loss in quiet of 39 dB would have on average 13 dB of traditionally incompensible loss. Also, Plomp (1978) observed that individual data differed considerably from the mean. This individual variability could be due to individually different degrees of and causes for the measured loss, or due to insufficient measurement accuracy. An important observation he made was that data points from listeners with age-related hearing loss agreed well with data points based from studies on other sensorineural hearing impairments. As a consequence he considered age-related hearing loss to be primarily due to deterioration in the auditory pathway rather than to mental impairment. This last point is fundamental considering that mental impairment can most likely not be compensated by signal processing.\n\nHowever, these findings have to be taken with care. The data used by Plomp (1978) were measured at different sites with different speech tests using different setups. The resulting list of possible systematic and random errors in the underlying data is long. The main contribution to the systematic error (expectable differences across studies) apart from the calibration error and the different listener panels was probably the use of different speech tests (including measurement paradigm, speech material, masker, presentation, ...). It is known, that the type of speech material (e.g., logatomes, numbers, isolated words, or sentences) makes a difference in the outcome of a speech in noise recognition experiment. Hence, tests with different speech material might also be differently susceptible to hearing loss and result in different values of SHLD. There is no reason to assume that SHLD is independent from the speech test. The main contribution to the random error (unpredictable differences across measurements) was probably different for the studies and due to the stochastic nature of the measurement procedures. Both, the random and the systematic errors were not specifically considered in the analysis of Plomp (1978). The variety of unknowns makes it difficult to translate his finding to expectable values and individual variability of SHLD with the employed matrix sentence test. Matrix sentence tests were designed to minimize the random error in the measurement of an SRT, where Kollmeier et al. (2015) reported a test-retest reliability of 0.5 dB for listeners with normal hearing and 0.7 dB for listeners with impaired hearing.\n\nFortunately, a suitable data set was measured with a matrix sentence test. Wardenga et al. (2015) found a similar relation between the degree of hearing loss and SHLD for the German matrix sentence test in the (stationary) test-specific noise condition, where the SRT in noise increased by about 1 dB every 10-dB increase in PTA5 for PTAs below 47 dB HL (Figure 5 there). This is much less than the average (1 dB every 3-dB) increase found by Plomp (1978). One reason for the lower increase could be that the speech hearing loss in quiet (SHLA+D) includes the speech hearing loss in noise (SHLD), while the PTA might not. But that alone cannot explain the huge difference. Another reason for the difference could be that Wardenga et al. (2015) derived the relation from individual data with relatively low PTAs while Plomp (1978) derived the relation from averaged data including all degrees of hearing impairment. It is probably fair to assume that the data from Wardenga et al. (2015) offers a more conservative and accurate estimate of the minimum expected average speech hearing loss in noise.\n\nTaking the relation from Wardenga et al. (2015) (which is based on data from 177 listeners aged 17 to 82y) as an orientation, it indicates that the average SHLD for PTAs of 60 dB HL could be about 6 dB in stationary noise. The individual variability of SHLD in their analysis was reported with 1.17 dB for PTAs below 47 dB HL, which was only twice the test-retest reliability of the matrix test. An interpretation of this relation could be that a given hearing loss in quiet is very likely related to a certain hearing loss in noise. This interpretation would be only valid if additional amplification would not change the relation, that is, if the same relation was observed for a noise presentation level of, e.g., 75 dB SPL instead of 65 dB SPL. However, it is difficult to speculate on that. On the one hand, the test-specific noise (OLNOISE) reduces the effect of the individual hearing threshold by masking the speech signals with a stationary matched-spectrum noise. On the other hand, for higher PTAs, the individual hearing threshold will eventually exceed the noise level at high frequencies, which could be compensated by amplification. Then again, according to the simulations presented in this contribution, the removal of high-frequency portions (>4000 Hz) of the signals in the OLNOISE condition had little effect on the SRT. These considerations demonstrate the inherently non-linear relations between the parameters assumed to affect speech recognition in noise, and hence the difficulty of abstracting these from a given context.\n\nNonetheless, to continue with an specific value for SHLD for the German matrix sentence test that could likely be found in the wild, the relation established by Wardenga et al. (2015) is assumed to ve valid. In favor of this assumption is that the linear regression estimated from the data with PTAs below 47 dB HL describes the data in their Figure 5 (solid black line) really well. One would expect an increase in variability with the PTA (below 47 dB HL) if the absolute hearing threshold affected the SRT, such as it was observed for higher PTAs; but this was not the case. Accordingly, Wardenga et al. (2015) concluded that with 65 dB SPL fixed noise presentation level the SRT is mainly determined by listening in noise for PTAs <≈47 dB HL, and above it is mainly determined by listening in quiet. Based on these considerations, it seems likely that listeners with an SRT of 0 dB SNR in the German matrix sentence test in the test-specific stationary noise exist whose speech recognition performance cannot be improved further by simple amplification. That would indicate an SHLD of about 7 dB. This estimate can be used as an orientation to interpret the right-most column in Table 1, which is equivalent to the SHLD. For example, profile P-2000-14 with an SHLD of 6.5 dB lies within the range of empirically observed values. In this context, profiles P-1000-14 and P-2000-21 might be border cases with values of 9 to 10 dB for SHLD, and profile P-1000-21 lies probably outside range of empirically observed values for SHLD. Profiles P-8000-21, P-4000-14, P-2000-14, and P-1000-7, are all compatible with a large SHLD of less than 7 dB in the stationary noise condition. The key difference between these profiles is the proportion of SHLD that is due to the level uncertainty and hence might be compensable by a PLATT expansion. Assuming that the average available frequency range to listeners with impaired hearing is between 2000 and 4000 Hz, P-4000-14 and P-2000-14 could represent parameter values in a realistic range.\n\nThe aim of classifying profiles into more and less realistic ones is to use them to formulate a quantitative hypothesis on the expected benefit in SRT due to PLATT expansion. This does not mean that the considered profiles exist. The assumption that the reduction in speech recognition performance in noise is due to a combination of a limited frequency range and a mechanism similar to an increased level uncertainty justifies their use. While this assumption may be sensible, it doesn’t mean that it is correct. This would have to be tested in listening experiments. For the effect of the limited frequency range, this could be achieved by, e.g., low-pass filtering the signals in speech recognition experiments. For the effect of an increased level uncertainty, a direct verification is currently not possible because the level uncertainty adds a noise in a domain which is not accessible for manipulations in experiments with human listeners; unlike for the limitation of the frequency range, there is no known equivalent signal manipulation. As explained in the Introduction, the most promising (and at the same time constructive) approach to test whether a mechanism similar to an increased level uncertainty causes a part of the class D hearing loss of listeners with impaired hearing is trying to compensate it.\n\nThe expansion feature of the PLATT dynamic range manipulation approach aims to mitigate the increase in SRT due to an increased level uncertainty. It was specifically designed to compensate the effect of the level uncertainty on speech recognition performance such as it is implemented in FADE. The presented simulation results showed that this necessary interim goal was achieved and indicate that about half of class D loss due to an increased level uncertainty was compensated. This only demonstrates that the expansion works as intended in the context of the model. There is no reason to assume that the results, that is, the partial compensation of a class D hearing loss, would transfer to experiments human listeners. Now—the key question of this contribution—what will happen in experiments with human listeners, in case the central model assumptions are incorrect?\n\n\n\nA) If the PLATT expansion does not improve the SRTs in noise of human listeners with a class D hearing loss, this would indicate that the mechanism that causes the class D loss is not well described by the level uncertainty, because the PLATT expansion interacts differently with the mechanism in human listeners than with the mechanism in the model.\n\nB) If the PLATT expansion improves the SRTs in noise of human listeners and if these improvements are in line with individual predictions, this would strongly support the hypothesis that a mechanism similar to an increased level uncertainty causes a part of the class D hearing loss of listeners with impaired hearing.\n\nC) However, more likely than B), if the PLATT expansion improves the SRTs in noise of human listeners but the improvements are found to be substantially smaller than predicted, this would only partly support the hypothesis that a mechanism similar to an increased level uncertainty causes a part of the class D hearing loss of listeners with impaired hearing.\n\nIn scenario A), a central model assumption is incorrect. The (to-be) collected empirical data still can help to improve the model, but the expansion feature of PLATT would be useless in the context of a hearing device. In scenario B), the central model assumptions are probably correct. The (to-be) collected empirical data cannot help to improve the model, but the expansion feature of PLATT would overcome a serious limitation of current hearing device technology. In scenario C), most of the central model assumptions were probably correct. The (to-be) collected empirical data can probably help to improve the model, and the expansion feature of PLATT could give strong hints regarding how to overcome a serious limitation of current hearing device technology. In this last scenario, a differentiated analysis of the predictions errors will be helpful in tracking down the inaccurate assumptions. The probabilities for scenario A), B), and C) are unknown. To provide evidence for any scenario, an experiment with human listeners suitable to unmistakably show the compensation effect has to be conceived.\n\nThe presented simulation results clearly show the potential of the expansion of spectral modulation patterns in the range between 2 and 4 ERB, as implemented in PLATT, to compensate a class D hearing loss that was implemented by an increased level uncertainty. In both noise conditions, approximately half of the class D loss due to an increased level uncertainty was compensated, while the class D loss due to a limited frequency range was not compensated. Narrowing down the simulation results to profiles P-4000-14 and P-2000-14, which were identified as the more realistic ones, the predicted improvements in SRT with PLATT-6 were 3.0 dB in the stationary noise condition. These improvements would correspond to a compensation of 3.04.9≈61% and 3.06.5−1.2≈57% of the class D hearing loss due to the increased level uncertainty. In the fluctuating noise condition, improvements in SRT of 4.6 and 5.3 dB were predicted with PLATT-6, with profiles P-4000-14 and P-2000-14, respectively. These improvements would correspond to a compensation of 4.611.4−3.2≈56% and 5.314.3−6.5≈68% of the class D hearing loss due to the increased level uncertainty. Hence, if the model assumptions are correct, benefits in SRT of about 3.0 dB and 5.0 dB in the stationary and fluctuating noise condition are expected, which correspond to more than 50% of the respective class D losses due to the level uncertainty. Such benefits would be individually measurable with the standard German matrix sentence test. However, according to the profile P-4000-7, for which benefits in SRT of up to 1.3 dB and 2.7 dB were predicted in the stationary and fluctuating noise condition, respectively, individual measurements would possibly not yield significant results. This is because the individual benefit in SRT is derived from two individual measurements (aided and unaided), each generating a random error of about 0.7 dB (standard deviation) for listeners with impaired hearing. If the real benefit is lower than about (0.7⋅2⋅1.96≈1.94≈) 2 dB, a significant (with p-value <0.05) effect can be only shown in group averages but not in single individual measurements. However, showing an effect in individual measurements is highly preferable because then the data could be used to further analyze the individual characteristics of listeners with and without benefits. Hence, even if this goal might not be achieved, experiments with human listeners should be designed with the aim to maximize the absolute effect.\n\nThe optimal (in terms of improvements in SRT) value for the expansion factor depended on the level uncertainty parameter. For lower values of the level uncertainty, in many conditions lower factors (4 or 6) resulted in the best speech recognition performance, but higher values did not decrease the improvement much. For the profiles P-4000-14 and P-2000-14, a factor of 8 was optimal. From this perspective, nothing speaks against evaluating PLATT with high expansion factors in listening experiments. Because an expansion factor of 8 results in a strong modification of the signal, and because it is unknown how such modifications are perceived in terms of quality and loudness by listeners with impaired hearing, at least two values for the expansion factor should be evaluated in listening experiments.\n\nIn measurements with human listeners, speech recognition performance, audio quality perception, and loudness perception all depend on the presentation level. In this contribution, efforts were made to separate the speech recognition performance from the presentation level as much as possible. This resulted in considering elevated presentation levels, and loudness perception is strongly related to presentation levels. Also in this contribution, efforts were made to mitigate audible artifacts in the manipulation and resynthesis stages of PLATT. But the non-linear manipulation inevitably results in audible artifacts which probably affect audio quality perception. The effect of processing noisy speech signals at 0 dB SNR with PLATT-1, PLATT-4, and PLATT-8 is illustrated in Figure 22. The spectro-temporal maxima of the shown log Mel-spectrograms are 68.8, 73.4, and 79.8 dB SPL for the noisy signals in stationary noise, and 77.0, 81.8, and 89.3 dB for the noisy signals in fluctuating noise. The corresponding effective amplitudes (RMS) are 66.6, 69.2, and 74.4 dB SPL for the noisy signals in stationary noise, and 69.7, 72.3, 77.8 dB for the noisy signals in fluctuating noise. The increase in RMS and maximum power, with PLATT-8 by about 8 dB and more than 10 dB, respectively, suggests an increased loudness perception and also demonstrates the necessity to evaluate the approach in conditions in which an increase in level must not result in an improvement in SRT. Also, the processing will probably have an effect on the perceived audio quality, where, with increased expansion factors, the audio quality will eventually decrease. In the subjective opinion of the author, based on a comparison of unprocessed and processed signals, the artifacts due to processing the stimuli with PLATT-1 are minor, with PLATT-4 clearly audible, and with PLATT-8 pronounced. While it would be a difficult task to predict the effect of the processing on the perception of audio quality of listeners with normal hearing, it would be even more so for listener with impaired hearing.\n\nIllustration of the effect of the expansion factor with PLATT: Log Mel-spectrograms of processed noisy speech in stationary (left column) and fluctuating noise (right column) at 0 dB SNR processed with PLATT-1 (top row), PLATT-4 (center row), and PLATT-8 (bottom row).\n\nApart from speech recognition performance, individual loudness and audio quality perception are important perceptual dimensions for hearing aid users. The expected increase in perceived loudness and a possible decrease in perceived audio quality are considered collateral side-effects of a signal manipulation with PLATT. These side-effects can either be tolerated or mitigated/compensated with further extensions or modifications of the PLATT approach. It is unclear how a listener with impaired hearing would trade speech recognition performance for audio quality or loudness in the context of PLATT expansion. To gather evidence about the relation of PLATT expansion, loudness perception, and audio quality perception, the considered expansion factors should cover a wide range of these side-effects. Hence an evaluation of speech recognition performance, audio quality, and loudness with PLATT expansion factors of 1, 4, and 8 is proposed. This will help to better estimate the suitability of the raw approach in the context of hearing aids and show which properties could be in the focus for possible future optimization.\n\nThere are several possibilities to optimize the expansion approach with respect to loudness perception and quality. For example, the expansion factor can be chosen to be frequency dependent. If the frequency regions in which the benefits are achieved by expansion do not fully overlap with the frequency regions where loudness perception is critical, there could be potential to favorably trade speech recognition performance against loudness. The expansion factor could be limited, or mapped otherwise non-linearly to only expand (the low) modulation amplitudes required to improve speech perception in low-SNR conditions while leaving (the already high) modulation amplitudes in high-SNR conditions unmodified. A detailed discussion of possible modifications is considered too hypothetical to be further elaborated in this contribution.\n\nBased on the presented simulations and considerations, the following experimental measurement conditions are proposed to test the presented hypothesis that a part of a class D hearing loss can be compensated by expanding spectral patterns in the range between 2 and 4 ERB in speech in noise recognition experiments with human listeners. Regarding the noise maskers, an evaluation with the test-specific noise (e.g. OLNOISE for the German matrix sentence test), the fluctuating ICRA5-250 noise, and, in addition, a competing voice masker, e.g., the International Speech Test Signal (ISTS; Holube et al., 2010) or its optimized version with limited pause durations, the International Female Fluctuating Masker (IFFM6 ), is proposed. The IFFM masker is interesting because it shares the speech modulation properties with the target speaker and usually results in very weak masking for listeners with normal hearing. It was not included in the objective evaluation with FADE because predictions with FADE for competing talker scenarios are known to be inaccurate; Schädler et al. (2018) reported the effect of the masker to be overestimated by approximately 10 dB.\n\nThe noise presentation levels ideally would be sufficiently high to compensate any hearing loss which is compensable by simple amplification. For the stationary noise condition, this would be easier to achieve than with the fluctuating noise conditions. As shown in left panel of Figure 2, even mild hearing loss profiles are expected to require very high presentation levels to maximize the compensation with simple linear amplification. However, the improvements in SRT at high presentation levels can be expected to be reasonably small. As a compromise, and to avoid measuring the whole Plomp-curve, a reference experiment with an increased level can be performed to test if the equivalent linear amplification would result in a similar speech recognition performance than using PLATT expansion. The observed increase in RMS level with PLATT-8 was about 8 dB. Adding a small margin, 10 dB amplification should result in higher RMS levels of noisy speech signals at 0 dB SNR than processing the same signals with PLATT-8. Hence, noise presentation levels of 70 and 80 dB SPL are proposed. The proposed noise presentation level is 70 dB SPL for aided and unaided measurements, while the noise presentation level of 80 dB SPL serves as an alternative to the aided measurement; one in which 10 dB amplification is applied. If the processing with PLATT expansion achieves better SRTs than with the 10 dB linear amplification, this would indicate the compensation of a class D hearing loss. However, the RMS level is only a rough proxy to loudness perception. Hence, in all conditions (70 dB SPL unaided, 70 dB SPL aided, 80 dB SPL unaided), the loudness perception of characteristic signals, e.g., speech in noise at 0 dB SNR, should be measured to check if the perceived loudness of the processed 70 dB SPL signals is really lower than the perceived loudness of the 80 dB SPL signals.\n\nTo further mitigate any benefits due to simple linear amplification at high presentation levels, all stimuli should be low-pass filtered to achieve an effective frequency range of 2000 Hz. This would limit the evaluation of a benefit due to PLATT expansion to the frequency range up to 2000 Hz; the central frequency region that listeners with impaired hearing typically can use for speech recognition. A focus on this frequency region could be advantageous in a first evaluation. The low-pass filtering would make a group of listeners with impaired hearing less heterogeneous, because larger differences across listeners are usually observed at frequencies above 2000 Hz. And the loudness perception of such low-pass filtered stimuli might also be more pleasant than their corresponding broadband variants.\n\nIn total, the proposed measurements comprise 18 conditions: Three for training, three unaided measurements at 70 dB SPL (one for each noise masker), three unaided measurements at 80 dB SPL (also one for each noise masker), and nine measurements with PLATT-1, PLATT-4, and PLATT-8 (that is, three for each noise masker). In all conditions, SRT, loudness perception and audio quality perception are recommended to be assessed.\n\nIn addition, it is recommendable to characterize the listeners in the way Schädler et al. (2020a) proposed it, that is, with tone detection and tone in noise detection experiments, and not only with clinical audiograms. This additional data could be used to perform individual predictions of benefits with FADE and compare them to the individual measurements. The comparison of individual measurements and predictions is crucial to detect invalid assumptions in the model.\n\nAs speech material for the SRT measurements, the optimized matrix test sentences in native language are recommended Kollmeier et al. (2015). These are phonetically balanced and optimized for a high test-retest reliability. To minimize the training effect, three training lists of 20 sentences in noise are recommended. Lists of 30 sentences obtain a higher test-retest reliability but can also result in excessively long measurements which can be fatiguing. Hence, as a compromise, measurements with lists of 20 sentences could be used and repeated on a different day, which would also allow to detect a possibly remaining training effect.\n\nFor a first approach, headphone measurements are preferable over free-field measurements because they can be performed monaurally, which is recommendable. A monaural presentation prevents the interference of possibly individual binaural effects and facilitates the comparison to model predictions, as discussed by Schädler et al. (2020a).\n\nOnce there is evidence whether the proposed compensation strategy has a positive effect on speech recognition performance of listeners with impaired hearing, the band-width limitation to 2000 Hz that was recommended for the first experiments should be removed. For the next steps, it would not be important anymore to demonstrate the compensation of a class D loss alone, but to show benefits in more realistic and individually optimized configurations. Hence, there should be a shift towards a joint individual optimization of the compensation of class A and class D loss, loudness perception, and audio quality. Then, care would need to be taken to individually normalize loudness perception to get meaningful data. If the expansion approach with PLATT proves to work in monaural listening conditions, the concept should be extended to a binaural listening condition. For this, a free-field setup with a mobile hearing aid prototype hardware is recommendable to correctly assess individual binaural hearing, including binaural loudness perception. To better understand which speech portions are most affected by PLATT expansion, it would also be interesting to study its effect on phonemic contrasts.\n\n\nConclusions\n\nThe most important findings of this work can be summarized as follows:\n\n• The functional modeling of the class D hearing loss with the framework for auditory discrimination experiments (FADE), implemented by means of the level uncertainty, was interpreted as the counterpart of a compensation strategy which aims to (partially) compensate a class D heaing loss.\n\n• The strict low-delay constraints in hearing aid applications only allow for a manipulation of mainly spectral modulation patterns. Of these, the patterns in the range of 2 to 4 ERB seem especially suitable to protect them against the effect of the level uncertainty by dynamic range expansion.\n\n• A low-delay, real-time capable implementation of a patented dynamic range manipulation scheme (PLATT) which allows to perform the required dynamic range expansion was proposed. The implementation was optimized to run in real-time on the Raspberry Pi 3 Model B platform.\n\n• The evaluation of the PLATT expansion with FADE for several idealized profiles of hearing loss indicated that approximately half of the class D hearing loss due to an increased level uncertainty was compensable.\n\n• Simulations with FADE were used to predict the outcomes of specific speech recognition experiments prior to performing these. The underling hypothesis, that a class D hearing loss can be (partially) compensated, can be directly tested in an experiment with human listeners in the same listening conditions. Recommendations for this experiment were elaborated.\n\n\nData availability\n\nUnderlying data\n\nZenodo: m-r-s/measurement-prediction-framework: Thoughts on the potential to compensate a hearing loss in noise, http://doi.org/10.5281/zenodo.4500810 (Schädler (2021))\n\nThis project contains the following files:\n\n• The modified feature extraction.\n\n• The reference implementation of PLATT and the used configuration files.\n\n• The scripts which prepare and run the FADE simulations using the modified feature extraction and the reference implementation of PLATT.\n\n• The scripts which evaluate raw the experimental results and plot the results figures.\n\nData and scripts are available under GNU General Public License.\n\nThe speech material of the German matrix sentence test and the corresponding test specific noise signal (OLNOISE) is available from: https://www.hoertech.de/en/devices/intma.html\n\nThe ICRA5-250 noise signal is avaliable from: http://medi.uni-oldenburg.de/download/ICRA/index.html\n\n\nSoftware availability\n\nFADE version 2.4.0 was used for the FADE simulations: https://doi.org/10.5281/zenodo.4003779 (Schädler & Hülsmeier (2020c)).\n\nThe Hidden Markov Toolkit (used by FADE) is available from: http://htk.eng.cam.ac.uk/", "appendix": "References\n\nBisgaard N, Vlaming MS, Dahlquist M: Standard audiograms for the IEC 60118-15 measurement procedure. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWagener K, Brand T, Kollmeier B: Entwicklung und Evaluation eines Satztests für die Deutsche Sprache I-III: Design, Optimierung und Evaluation des Oldenburger Satztests. Zeitschrift für Audiologie 1999; 38(1-3): 4–15.\n\nWagener KC, Brand T, Kollmeier B: The role of silent intervals for sentence intelligibility in fluctuating noise in hearing-impaired listeners. Int J Audiol. 2006; 45(1): 26–33. PubMed Abstract \| Publisher Full Text\n\nWardenga N, Batsoulis C, Wagener KC, et al.: Do you hear the noise? The German matrix sentence test with a fixed noise level in subjects with normal hearing and hearing impairment. Int J Audiol. 2015; 54(sup2): 71–79. PubMed Abstract \| Publisher Full Text\n\nYund EW, Buckles KM: Multichannel compression hearing aids: Effect of number of channels on speech discrimination in noise. J Acoust Soc Am. 1995; 97(2): 1206–1223. PubMed Abstract \| Publisher Full Text\n\n\nFootnotes\n\n1 https://github.com/m-r-s/hearingaid-prototype.\n\n2 https://doi.org/10.5281/zenodo.4394186.\n\n3 https://doi.org/10.5281/zenodo.4003779.\n\n4 For example: https://github.com/m-r-s/hearingaid-prototype or https://batandcat.com/portable-hearing-laboratory-phl.html.\n\n5 pure-tone average.\n\n6 https://www.ehima.com/documents/." }	[ { "id": "83763", "date": "17 May 2021", "name": "Jon P Barker", "expertise": [ "Reviewer Expertise Automatic speech recognition", "machine listening" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper considers hearing impairment as a sum of loss due to attenuation and loss due to distortion, as proposed by Plomp (1978). It considers, in particular, the contribution of the distortion component (modelled as internal noise) and asks how much the deficit caused by this component might be restored by well-designed dynamic range manipulation. A patented dynamic range manipulation scheme (PLATT) is presented which works by decomposing spectral modulations into different frequency bands and applying a compression scheme that treats each band separately. The benefit of PLATT for ameliorating distortion loss is then tested by measuring the predicted SRT-50 improvements for speech in different noise conditions and listeners with different hearing profiles (where distortion loss if modelled by adding level uncertainty to the spectro-temporal signal representation). These experiments have used conditions that isolate the effect of distortion loss (i.e., comparing conditions with identical attenuation loss). The FADE (ASR-based) model is used to predict listener behaviour.\nThe author is clear to point out that the purpose of the paper is not to make specific claims for the benefit of PLATT in real listening conditions, but instead, to make specific and testable predictions about the outcomes of (yet-to-be-performed) listening experiments. In particular, the experiments will test the hypothesis that the distortion component of hearing loss can be modelled by level-uncertainty. The paper concludes by discussing the design of the listening studies and by presenting thoughts on the future development and evaluation of PLATT in real-world listening conditions where both distortion and attenuation need to be considered.\nI greatly enjoyed reading this well-written, detailed and thought-provoking paper. I have no major criticisms but have listed a few points for clarification below and a number of specific typos that can be fixed.\nFor clarification\nOn page 19, the paper says \"the curves show some variability which is due to the stochastic nature of the simulation process.\" I was not quite sure how to interpret this. Is this stochasticity in the FADE model, e.g. in the ASR training and testing? If so could the variability in the result be reduced by averaging over a greater number of simulated runs. If not would it be useful to add error bars to the Plomp plots.\n\nOn page 26, the hypothesis and outcomes of the future listening experiments are very clearly stated, and it is anticipated that the PLATT expansion may improve SRTs by less than predicted. It is said that this will be taken to mean that increased level uncertainty causes only a part of the class D hearing loss. However, could there be other reasons why listeners would find it hard to process the PLATT signals. For example, they might successfully reduce uncertainty but produce signals that do not match their expectations of speech signals, i.e., in FADE the ASR system is trained extensively on the processed signal, but in the listening experiments, listeners have their own internal speech models. Would listeners need extensive listening to adapt to the signals?\n\nOn page 29 it is noted that FADE is a poor predictor of SRTs for speech in the presence of speech interferers. I think it would be interesting to say a little about why this might be the case. Is it that it does not explicitly model informational masking effects, or that it does not model source separation? And if so are these factors that could be at play in the processing of the speech plus ICRAS-250 noise? And could these factors interact with the PLATT processing? e.g., cues for source separation being compromised by the non-linear PLATT processing.\n\nSpecific points and typographical errors,\nPage 3,\"This limit is still way above\" -> \"This limit is still far above\" (more formal and more easily understood)\nPage 8, end of first paragraph, \"be performed human listeners\" -> \"be performed by human listeners\"\nPage 10, \"fewer frequency channels reduce\" -> \"using fewer frequency channels reduces\"\nPage 11, \"raises the question which representation\" -> \"raises the question of which representation\"\nPage 11, Figure 5, please remake with larger text\nPage 12, after eq (1), \"and f_b the sampling frequency\" -> \"and f_s the sampling frequency\" (or change f_s to f_b in the equation)\nPage 13, Spectro-temporal signal representation: the envelope estimate would be clearer if presented with an equation.\n\nPage 16, after 1760 at end of page add, (2 x 11 x 16 x 5) just to be clear about how the total number of conditions arises.\nPage 18, line 2, \"as one might have expect.\" -> \"as one might have expected.\"\nPage 18, start of final paragraph, \"present effect of\" -> \"present the effect of\"\nPage 19, paragraph 2, \"due to PLATT in was\" -> \"due to PLATT was\"\nPage 25, final paragraph, \"with an specific\" -> \"with a specific\"\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "96389", "date": "18 Oct 2021", "name": "Arne Leijon", "expertise": [ "Reviewer Expertise Probabilistic pattern recognition", "model-based signal processing", "hearing technology", "psycho-acoustics." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMain Comments\nThis is a good paper. I agree with the author that it contributes to the scientific topic that may rightly be called “theoretical Audology”:\nThe paper summarizes several earlier studies indicating that the specific difficulty of people with impaired hearing to understand speech in noise is caused by a reduced information-transmission capacity of the peripheral auditory system (\"class D\" loss). These earlier results also suggest that the information loss has similar effects as an artificially induced spectro-temporal level uncertainty in the front-end processor of a simulation system for auditory discrimination (FADE), which uses an automatic speech recognition system to quantify the effects on speech recognition.\nConsidering the hypothesis that the level uncertainty in the computational auditory model might correspond to some physiological processes in the auditory system of real people, the author has designed a signal-processing system (PLATT) that can be used in real hearing aids to compensate for the level uncertainty, by enhancing spectral modulation patterns that are most important to convey phonetic contrasts in real speech.\nThe paper clearly explains the main features of the PLATT system, and presents simulation results showing that about half of the simulated loss caused by the level uncertainty could be compensated by hearing-aid spectral expansion. Of course, the results from model simulations in a computer might be seen as just an advanced form of mathematical speculation, and the author wisely emphasises that these simulation results cannot prove that PLATT is beneficial for real listeners.\nInstead, the paper uses the simulation results as a starting point to discuss how an empirical speech recognition experiment should be designed to be able to test the two hypotheses, (1) that something like the modelled level uncertainty causes the speech-recognition loss in real listeners, and (2) that spectral modulation enhancement in a hearing aid can (at least partly) compensate for this loss.\nThe paper shows that it is far from trivial to design such an experiment with real listeners. The experiment should separate the specific effects of modulation expansion from the effects of linear (or slow-compression) amplification, because both types of processing can have effects on speech recognition, loudness, and subjective quality of the processed speech in noise. Another serious difficulty is the test-retest uncertainty of all speech-recognition test methods.\nI think all of this discussion is scientifically valuable and should be approved for indexing. I have only a few suggestions to make the presentation easier to read and understand. (I have not studied the code for the FADE and the PLATT system, but I think I have understood the approach and I appreciate that the code has been made publicly available.)\nSuggestions for Clarity:\nTemporal modulation patterns: The second point in the Conclusions states that hearing aids “… only allow for a manipulation of mainly spectral modulation patterns.”\nThis formulation seems a little too strong: All compression systems have some effects on the temporal “modulation patterns” of the input signal. For example, slow automatic gain control (AGC) (with long attack time and long release time) tends to compress only the very slow temporal modulations while leaving faster modulations nearly unchanged. People have also studied AGC systems controlled by several signal features in parallel, with different time constants, with more complex effects on the temporal modulation patterns. The proposed PLATT system also “manipulates” the temporal “modulation patterns”, although it was not specifically designed for that purpose.\nFigure Captions and Table Header: I guess I am not the only reader tempted to look at the figures before reading the detailed explanations in the surrounding text. I understand the figure caption can not include a long complicated explanation, but, to facilitate understanding, the figure captions should at least define all graphical elements seen in the figure. No problem if the caption gets long.\nExamples:\nFigure 9: This figure is complicated but very important for readers to understand, as it is central for the explanation of PLATT. The caption should clearly state that the labels 1,2,3,4 refer to the differences between the displayed curves, because text labels in a graph most often refer to the plotted curves, not to the distance between curves. Perhaps this could be even more clearly shown graphically by a vertical line indicating the difference, connected to the arrow line to the label.\nAlso, at first sight, I was confused by the use of grayscale: The lightest gray curve in the GAIN plot is not the difference between the correspondingly light-gray curves in the OUTPUT and the INPUT plots, which would be the default interpretation. Instead, a gray GAIN curve is the difference between the gray OUTPUT (partial) spectrum and the solid black curve for the total INPUT spectrum. This should be explained in the caption. I spent some time searching the text before I found this explanation.\nFigure 10: The caption text seems to suggest that there are three spectra with only pure tones, and another three spectra with added white noise, but there is only one dotted curve, showing the spectrum of the pure tones with peaks about 60 dB SPL with added white noise. (In this figure, the gray GAIN curves are actually the difference between similarly gray OUTPUT and INPUT spectra, contrary to the use of gray-scale in Fig 9.)\nFigures 12-18: The second and fourth plot titles state “difference in SRT”, but the plot actually shows a positive “difference in SRT”, i.e., an increased SRT as a negative number, because it indicates a decrease in performance. This is of course easy to understand, but should still be mentioned in the first caption. Also, the meaning of legend text strings, like “P-8000-1 none” must be explained in the caption. At first sight of Fig 12, I could not guess the intended meaning of “none”.\nTable 1: The column with heading “NH” should be explained more clearly in the table header. The numerical values in this column do actually not indicate “the normal-hearing listener profile” as stated. The numbers show the difference between results for the NH and the unaided impaired profile on the same row in the first column. I needed to read the explanation on the following page before I understood that the numbers in the “NH” column are meant to be interpreted as ideal measures to which the benefit measures shown in the “PLATT-x” columns might be compared.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-311
https://f1000research.com/articles/10-310/v1	22 Apr 21	{ "type": "Review", "title": "Optical coherence tomography’s current clinical medical and dental applications: a review", "authors": [ "Saqib Ali", "Saqlain Bin Syed Gilani", "Juzer Shabbir", "Khalid S. Almulhim", "Amr Bugshan", "Imran Farooq", "Saqib Ali", "Saqlain Bin Syed Gilani", "Juzer Shabbir", "Khalid S. Almulhim", "Amr Bugshan" ], "abstract": "Optical coherence tomography (OCT) is a non-invasive investigative technique that is used to obtain high-resolution three-dimensional (3D) images of biological structures. This method is useful in diagnosing diseases of specific organs like the eye, where a direct biopsy cannot be conducted. Since its inception, significant advancements have been made in its technology. Apart from its initial application in ophthalmology for retinal imaging, substantial technological innovations in OCT brought by the research community have enabled its utilization beyond its original scope and allowed its application in many new clinical areas. This review presents a summary of the clinical applications of OCT in the field of medicine (ophthalmology, cardiology, otology, and dermatology) and dentistry (tissue imaging, detection of caries, analysis of dental polymer composite restorations, imaging of root canals, and diagnosis of oral cancer). In addition, potential advantages and disadvantages of OCT are also discussed.", "keywords": [ "Optical coherence tomography", "OCT", "Imaging", "Medicine", "Dentistry" ], "content": "Introduction\n\nOptical coherence tomography (OCT) is a non-invasive diagnostic method that can be utilized to acquire high-resolution three-dimensional (3D) images of a biological structure.1 OCT has quickly gained popularity among clinicians due to several advantages that include delivering quality images with quick imaging speed.2 This technique has several clinical applications that include (but are not limited to) detection of diabetic macular edema,3 epithelial mapping for refractive surgery,4 diagnosing and managing patients with neuro-ophthalmic conditions,5 imaging of retinal microcirculation to detect hemodynamic disturbances,6 detection of age associated changes in teeth,7 diagnosing dental caries8 and tooth wear,9 and post-treatment assessments in dentistry.10\n\nThe aim of this review is to summarize the clinical applications of OCT in the field of medicine and dentistry, and highlight its potential advantages and disadvantages. Google Scholar and PubMed databases were searched using keywords including “optical coherence tomography,” “OCT,” and “OCT and its clinical applications.” Our search revealed 500+ hits; all the articles published in languages other than English and conference abstracts were excluded. Only the relevant articles published in the last 10 years (2011-2021) were included to keep the information recent and contemporary. Finally, 83 articles were selected for this study and included in our review.\n\n\nClinical applications of OCT\n\nOCT has been utilized by medical professionals extensively for diagnostic purposes and has several medical applications in ophthalmology, cardiology, otology, and dermatology. These applications will now be discussed below.\n\nOphthalmology\n\nThis technique can be used to accurately analyze choroidal thickness in the eyes under conditions such as diabetic neuropathy and macular degeneration due to aging.11 In fact, it can be said with authority that this imaging tool is nowadays a routine part of ophthalmology practice to identify macular lesions.12 The role of OCT is also pivotal in detecting other eye-related conditions like hyper-reflectivity due to macular telangiectasia type-2,13 optic neuropathies,14 and glaucomatous retinal nerve fiber layer loss.15 Papilledema is a serious medical condition that leads to visual disturbances and occurs due to the optic disc's swelling because of increased intraocular pressure;16,17 OCT technique can be used to measure the amount of optic disc edema in this condition.18\n\nCardiology\n\nOCT can be used for various applications in the field of cardiology. In a recent study, it has been reported that OCT-based fractional flow reserve learning can be used for enhanced treatment of intermediate coronary artery stenosis.19 This technique can also be used for coronary imaging,20 guiding percutaneous coronary intervention,21 diagnosing myocardial infarction with non-obstructive coronary arteries,22 and observing healed coronary plaques.23\n\nOtology\n\nOCT can be used to accurately diagnose otology conditions such as otitis media and conductive hearing loss.24 Other otology conditions where OCT has been used in the literature include diagnosis of middle ear effusions,25 visualizing middle ear exudate,26 observing microstructures of the middle and inner ear through an extra tympanic approach,27 visualization of intra-cochlear structures for future cochlear implant surgery,28 and to study endolymphatic hydrops that develops due to noise trauma.29\n\nDermatology\n\nOCT has the potential to be used to image skin layers and associated structures. In a recent study, Kato et al. utilized OCT to measure sweating and reported that this technique could be used successfully to measure and quantify sweating in the body.30 Other OCT uses related to dermatology include characterization of micro-environment of facial pores,31 aiding in the diagnosis of basal cell carcinoma,32 observing the treatment response of inflammatory dermatoses,33 and to detect non-melanoma skin cancers.34\n\nIn the past, the use of OCT was only limited to the assessment of dental hard and soft tissue morphology; however, as knowledge related to the mechanics and use of OCT increased, it led to more widespread use of OCT for different objectives. Nowadays, OCT is being used in various clinical and research applications related to dentistry. The uses of OCT in the field of dentistry are discussed in detail below.\n\nTissue imaging\n\nMany dental tissues such as enamel and dentin can be easily visualized utilizing OCT.35 The optical characteristics of enamel and dentin are different, so these structures can be distinguished from each other. Hariri et al. reported that OCT signals in dentin vary due to the presence of dentinal tubules and are also different from enamel.36 The direction of the OCT beam to the examined surface can affect the signal intensity. The sharp slope of the enamel surface may appear bright compared to the less steep surface.37 This technique has also been applied previously in various studies to detect the presence of enamel cracks.37,38 Imai et al. visualized enamel cracks utilizing OCT and demonstrated their extension beyond dentin-enamel junction.38 To detect tooth fracture in the coronal region, swept-source OCT (SS-OCT) is a useful technique.39\n\nDetection of dental caries and demineralization of teeth\n\nThere has been a paradigm shift in caries management recently, and this change indicates that caries should be managed conservatively.40 However, it requires detection of early, un-noticeable caries activity at the earliest for it to be truly conservative. Radiography is an unreliable method of detection of carious activity. Moreover, radiography cannot distinguish properly between active and arrested lesions.41 In an earlier study, it was reported that when physicians used OCT, they could detect tooth volumetric and thickness changes earlier compared with other conventional methods.42 Detecting initial changes in the tooth structure could be useful in hindering the progress of dental caries. In another study, the OCT technique effectively observed the depths of carious demineralization of dentin.43 Tooth demineralization can be differentiated from healthy tooth tissue by increasing light scattering in porous demineralized tooth tissue while performing OCT.44 SS-OCT possesses an advantage in cases where higher resolution and penetration depth are required for cavitated caries or deeper lesion detection.45 In enamel caries, the images appear brighter on grayscale OCT, and this could be the result of increased brightness due to the light reflection occurring between two homogenous structures with different refractive indices.46 The demineralized mineral crystals and water in the pores cause increased reflectivity resulting in characteristic brightness in the OCT image.46 SS-OCT can image dentin caries as a continuous bright area that extends from enamel into the dentin.44 The signal attenuation is considered as an object parameter to differentiate between sound and demineralized enamel.47 However, this attenuation can be affected by the wavelength of the incident light. The wavelength suggested to be used to detect demineralization is 1310-nm.48\n\nAnalysis of defective dental polymer composite restorations\n\nConventional OCTs are considered useful in detecting the cavitated border, fracture lines, and interfacial gaps in the tooth-restoration interface.49 This property appears to be due to strong reflection from the material surface in conventional OCT setups for detecting the reflective surface from the defects.50 Concerning dental polymer composites, the polymer's composition could affect signal attenuation of OCT and depends on the refractive index difference between polymers resin and the filler.51 OCT imaging can detect the air bubbles or void within the composite restoration52 and evaluate bonding interface.53 This assessment becomes easy as poorly sealed polymer composites demonstrate brightly clustered images (revealing gaps), whereas tightly fit boundaries do not exhibit too much scattering.54 Ishibashi et al. performed a study previously and reported that SS-OCT could detect resin-based polymer composite restorations defects as it provides a much higher resolution than other conventional techniques.55 The OCT technique is also a useful tool to analyze volumetric polymerization shrinkage (VPS). Sampio et al. performed a study to measure the VPS of different polymer composites via micro-computed tomography, and to qualitatively compare the gap formation through OCT.56 It was concluded from the results of their study that while both techniques were useful, VPS assessment was largely material dependent.56\n\nImaging of root canals\n\nIn an earlier in-vitro study, Lino et al. reported that SS-OCT could be used to locate the second mesiobuccal canal in maxillary molars.57 Rashed et al. also reported similar findings in their study and reported that SS-OCT has the potential to accurately measure residual dentin thickness (RDT) and image root canals along with other internal tooth anatomic structures like pulp horns and isthmus.58 The SS-OCT technique is a useful tool to accurately observe fractures of tooth roots and root canal endoscopy.59 Krause et al. utilized OCT to monitor the pulp chamber’s roof and RDT and reported that this technique could be useful in preserving the pulp’s vitality while preparing deep cavities in dentin.60\n\nDetection of oral cancer\n\nOral cancer is one of the most fatal and prevalent cancers in the world. In India, it is the third most typical form of cancer,61 and among those, 90% are oral squamous cell carcinomas.62 Lee et al. earlier reported that OCT is a reliable technique to diagnose oral precancerous lesions with high sensitivity and specificity.63 OCT has been successfully applied previously to diagnose oral malignancies in the hamster cheek pouch model.64 Adegun et al. used OCT to diagnose epithelial dysplasia and reported that this technique could offer non-invasive method to locate the appropriate site of biopsy and thus could be useful in the early diagnosis of oral cancer.65\n\nOther dental uses\n\nPeri-implantitis is the inflammation of structures around the implant, which may lead to bone resorption around the implant and eventual failure of the implant.66 OCT can detect and prevent peri-implant diseases.67 Kim et al. used OCT to measure peri-implant bone defects and reported that OCT was useful in assessing peri-implant bone levels and identifying bone defects.68 Luca et al. used OCT to analyze bone the bone following low-level laser therapy and reported that OCT could quantitatively measure bone regeneration.69 OCT is also a reliable tool to diagnose dental erosion,70 assess sealing performance of resin cements,53 detect invisible internal fractures of dentures non-invasively,71 and monitor resin-dentin gaps.72\n\n\nAdvantages and disadvantages of OCT\n\nOCT is a novel technique with diagnostic abilities used currently in different medical fields. High-resolution image acquisition of the biological tissue has enabled this modern technology to embed itself in the field of biomedical science, where researchers have been testing it for maximum possible utilization of its potential. The advantages and disadvantages of OCT have been summarized in Tables 1 and 2, respectively.\n\n\nFuture clinical applications\n\nConsidering the potential benefits of OCT, it can be predicted that its use in the medical field will increase further in the coming years. The combination of OCT with other techniques like scanning laser ophthalmoscopy (SLO) could prove to be useful for eye imaging when compared with the former approach alone.1 The use of OCT angiography (OCTA) technique to observe human vasculature could increase in the future80 as unlike SLO-based angiography methods; OCTA can provide depth information and isolation of vascular retinal81 and various choroidal layers.82 It will be interesting to discover the role of artificial intelligence (AI) while using OCT to diagnose skin diseases, particularly basal cell carcinoma.33 Deep learning algorithms have been applied previously to the OCT technique,83 but AI's role in improving OCT has not yet gained clinical maturity. It would be exciting to see the part of AI-related to OCT in the future, as it can decrease the cost and time required to train an operator.33\n\n\nConclusions\n\nOCT is a non-invasive technique that has several clinical applications and distinct advantages. It is an explorative diagnostic method that provides clinically pertinent data, is fast, and thus it is appropriate for research and clinical practice. OCT is still an emerging technology, and considering its potential benefits, its use in medical and dental clinics should be encouraged.\n\n\nData availability\n\nNo data is associated with this article.", "appendix": "References\n\nPodoleanu AG: Optical coherence tomography. J. Microsc. 2012; 247(3): 209–219. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIbne MM: Optical Coherence Tomography: Basic Concepts and Applications in Neuroscience Research. J Med Eng. 2017; 2017: 3409327. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGurreri A, Pazzaglia A: Diabetic Macular Edema: State of Art and Intraocular Pharmacological Approaches. Adv Exp Med Biol. 2021; 1307: 375–389. PubMed Abstract \| Publisher Full Text\n\nKhamar P, Rao K, Wadia K, et al.: Advanced epithelial mapping for refractive surgery. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNovozhilov AA, Shilyagin PA, Abubakirov TE, et al.: Non-contact optical coherence tomography - an effective method for visualizing the exudate of the middle ear. Vestn Otorinolaringol. 2020; 85(4): 16–23. PubMed Abstract \| Publisher Full Text\n\nOh SJ, Lee IW, Wang SG, et al.: Extratympanic Observation of Middle and Inner Ear Structures in Rodents Using Optical Coherence Tomography. Clin Exp Otorhinolaryngol. 2020; 13(2): 106–112. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStarovoyt A, Putzeys T, Wouters J, et al.: High-resolution Imaging of the Human Cochlea through the Round Window by means of Optical Coherence Tomography. Sci. Rep. 2019; 9(1): 14271. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBadash I, Applegate BE, Oghalai JS: In Vivo Cochlear imaging provides a tool to study endolymphatic hydrops. J Vestib. Res. 2020. 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PubMed Abstract \| Publisher Full Text\n\nImai K, Shimada Y, Sadr A, et al.: Noninvasive cross-sectional visualization of enamel cracks by optical coherence tomography in vitro. J Endod. 2012; 38(9): 1269–1274. PubMed Abstract \| Publisher Full Text\n\nFried WA, Simon JC, Lucas S, et al.: Near-IR imaging of cracks in teeth. Proc SPIE Int Soc Opt Eng. 2014; 8929: 89290Q. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlhussain AM, Alhaddad AA, Ghazwi MM, et al.: Remineralization of artificial carious lesions using a novel fluoride incorporated bioactive glass dentifrice. Dent Med Probl. 2018; 55(4): 379–382. PubMed Abstract \| Publisher Full Text\n\nGomez J: Detection and diagnosis of the early caries lesion. BMC Oral Health. 2015; 15: S3. 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PubMed Abstract \| Publisher Full Text\n\nHariri I, Sadr A, Nakashima S, et al.: Estimation of the enamel and dentin mineral content from the refractive index. Caries Res. 2013; 47(1): 18–26. PubMed Abstract \| Publisher Full Text\n\nMandurah MM, Sadr A, Shimada Y, et al.: Monitoring remineralization of enamel subsurface lesions by optical coherence tomography. J Biomed Opt. 2013; 18(4): 046006. PubMed Abstract \| Publisher Full Text\n\nWada I, Shimada Y, Ikeda M, et al.: Clinical assessment of non carious cervical lesion using swept-source optical coherence tomography. J Biophotonics. 2015; 8(10): 846–854. PubMed Abstract \| Publisher Full Text\n\nMakishi P, Shimada Y, Sadr A, et al.: Non-destructive 3D imaging of composite restorations using optical coherence tomography: marginal adaptation of self-etch adhesives. J Dent. 2011; 39(4): 316–325. PubMed Abstract \| Publisher Full Text\n\nShimada Y, Sadr A, Sumi Y, et al.: Application of Optical Coherence Tomography (OCT) for Diagnosis of Caries, Cracks, and Defects of Restorations. Curr Oral Health Rep. 2015; 2(2): 73–80. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLammeier C, Li Y, Lunos S, et al.: Influence of dental resin material composition on cross-polarization-optical coherence tomography imaging. J Biomed Opt. 2012; 17(10): 106002. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNazari A, Sadr A, Shimada Y, et al.: 3D assessment of void and gap formation in flowable resin composites using optical coherence tomography. J Adhes Dent. 2013; 15(3): 237–43. PubMed Abstract \| Publisher Full Text\n\nTurkistani A, Sadr A, Shimada Y, et al.: Sealing performance of resin cements before and after thermal cycling: evaluation by optical coherence tomography. Dent Mater. 2014; 30(9): 993–1004. PubMed Abstract \| Publisher Full Text\n\nTurkistani A, Nakashima S, Shimada Y, et al.: Microgaps and demineralization progress around composite restorations. J Dent Res. 2015; 94(8): 1070–1077. PubMed Abstract \| Publisher Full Text\n\nIshibashi K, Ozawa N, Tagami J, et al.: Swept-source optical coherence tomography as a new tool to evaluate defects of resin-based composite restorations. J Dent. 2011; 39(8): 543–548. PubMed Abstract \| Publisher Full Text\n\nSampaio CS, Arias JF, Atria PJ, et al.: Volumetric polymerization shrinkage and its comparison to internal adaptation in bulk fill and conventional composites: A μCT and OCT in vitro analysis. Dent Mater. 2019; 35(11): 1568–1575. PubMed Abstract \| Publisher Full Text\n\nIino Y, Ebihara A, Yoshioka T, et al.: Detection of a second mesiobuccal canal in maxillary molars by swept-source optical coherence tomography. J Endod. 2014; 40(11): 1865–1868. PubMed Abstract \| Publisher Full Text\n\nRashed B, Iino Y, Komatsu K, et al.: Evaluation of root canal anatomy of maxillary premolars using swept-source optical coherence tomography in comparison with dental operating microscope and cone beam computed tomography. Photomed Laser Surg. 2018; 36(9): 487–492. PubMed Abstract \| Publisher Full Text\n\nQi LY, Chen C, Jiang L, et al.: Construction of swept source optical coherence tomography imaging system for root canal endoscopy and application in diagnosis of root fractures. Beijing da Xue Xue Bao Yi Xue Ban. 2019; 51(4): 753–757. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKrause F, Köhler C, Rüger C, et al.: Visualization of the pulp chamber roof and residual dentin thickness by spectral-domain optical coherence tomography in vitro. Lasers Med Sci. 2019; 34(5): 973–980. PubMed Abstract \| Publisher Full Text\n\nSharma S, Satyanarayana L, Asthana S, et al.: Oral cancer statistics in India on the basis of first report of 29 population-based cancer registries. J Oral Maxillofac Pathol. 2018; 22(1): 18–26. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBugshan A, Farooq I: Oral squamous cell carcinoma: metastasis, potentially associated malignant disorders, etiology and recent advancements in diagnosis. F1000Res. 2020; (9): 229. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee CK, Chi TT, Wu CT, et al.: Diagnosis of oral precancer with optical coherence tomography. Biomed Opt Express. 2012; 3(7): 1632–1646. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPande P, Shrestha S, Park J, et al.: Automated classification of optical coherence tomography images for the diagnosis of oral malignancy in the hamster cheek pouch. J Biomed Opt. 2014; 19(8): 086022. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAdegun OK, Tomlins PH, Hagi-Pavli E, et al.: Quantitative analysis of optical coherence tomography and histopathology images of normal and dysplastic oral mucosal tissues. Lasers Med Sci. 2012; 27(4): 795–804. PubMed Abstract \| Publisher Full Text\n\nPrathapachandran J, Suresh N: Management of peri-implantitis. Dental Res J. 2012; 9(5): 516–521. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSanda M, Shiota M, Imakita C, et al.: The effectiveness of optical coherence tomography for evaluating peri-implant tissue: A pilot study. Imagin Sci Dent. 2016; 46(3): 173. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKim S, Kang SR, Park HJ, et al.: Quantitative measurement of peri-implant bone defects using optical coherence tomography. J Periodontal Implant Sci. 2018; 48(2): 84–91. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLuca RE, Todea CD, Duma VF, et al.: Quantitative assessment of rat bone regeneration using complex master-slave optical coherence tomography. Quant Imaging Med Surg. 2019; 9(5): 782–798. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAttin T, Wegehaupt FJ: Methods for assessment of dental erosion. Monogr Oral Sci. 2014; 25: 123–142. PubMed Abstract \| Publisher Full Text\n\nSumi Y, Ozawa N, Nagaosa S, et al.: Application of optical coherence tomography (OCT) to nondestructive inspection of dentures. Arch Gerontol Geriatr. 2011; 53(2): 237–241. PubMed Abstract \| Publisher Full Text\n\nBakhsh TA, Sadr A, Shimada Y, et al.: Non-invasive quantification of resin–dentin interfacial gaps using optical coherence tomography: Validation against confocal microscopy. Dent Mater. 2011; 27(9): 915–925. PubMed Abstract \| Publisher Full Text\n\nChang CJ, Huang YM, Hsieh MH, et al.: Flow signal change in polyps after anti-vascular endothelial growth factor therapy. PloS One. 2020; 15(10): e0241230. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBayer A, Akman A: Artifacts and Anatomic Variations in Optical Coherence Tomography. Turk J Ophthalmol. 2020; 50(2): 99–106. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMirzaei N, Shi H, Oviatt M, et al.: Alzheimer’s retinopathy: seeing disease in the eyes. Front Neurosci. 2020; 14: 921. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHagag AM, Gao SS, Jia Y, et al.: Optical coherence tomography angiography: technical principles and clinical applications in ophthalmology. Taiwan J Ophthalmol. 2017; 7(3): 115–129. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChopra R, Wagner SK, Keane PA: Optical coherence tomography in the 2020s—outside the eye clinic. Eye (Lond). 2020; 9: 1–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMaloney R: The Optovue iVue OCT System from Grafton Optical: the possibilities of hand-held OCT devices in ophthalmic practice. J Vis Commun Med. 2012; 35(2): 76–81. PubMed Abstract \| Publisher Full Text\n\nIsraelsen NM, Petersen CR, Barh A, et al.: Real-time high-resolution mid-infrared optical coherence tomography. Light: Sci Appl. 2019; 8(1): 1–3. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPoddar R, Migacz JV, Schwartz DM, et al.: Challenges and advantages in wide-field optical coherence tomography angiography imaging of the human retinal and choroidal vasculature at 1.7-MHz A-scan rate. J Biomed Opt. 2017; 22(10): 106018. PubMed Abstract \| Publisher Full Text\n\nChoi W, Mohler KJ, Potsaid B, et al.: Choriocapillaris and choroidal microvasculature imaging with ultrahigh speed OCT angiography. PloS one. 2013; 8(12): e81499. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKurokawa K, Liu Z, Miller DT: Adaptive optics optical coherence tomography angiography for morphometric analysis of choriocapillaris. Biomed Opt express. 2017; 8(3): 1803–1822. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDe Fauw J, Ledsam JR, Romera-Paredes B, et al.: Clinically applicable deep learning for diagnosis and referral in retinal disease. Nat Med. 2018; 24(9): 1342–1350. PubMed Abstract \| Publisher Full Text" }	[ { "id": "83913", "date": "27 Apr 2021", "name": "Farooq Ahmad Chaudhary", "expertise": [ "Reviewer Expertise Dentistry", "Oral Health", "and Preventive Dentistry" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article deals with the clinical applications of optical coherence tomography. It's well-written and I just have few minor comments:\nIntroduction could be expanded to include basics of the OCT technique.\n\nAn image showing the working principle of the OCT technique could be useful in the introduction section.\n\nIt would be easier for the readers if the article contained a flowchart of the search strategy.\n\nDental applications are well-written, but a little more explanation of medical applications could attract more readers.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [] }, { "id": "83915", "date": "29 Apr 2021", "name": "A. Thirumal Raj", "expertise": [ "Reviewer Expertise Oral pathology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article is interesting and summarizes various medical and dental applications of the optical coherence tomography (OCT) technique. I have reviewed it and would like to have it indexed. Few suggested improvements from my side are as follows:\nThe introduction section can be expanded further.\n\nMore medical applications of the OCT technique can be included.\n\nFuture work section can be more detailed.\n\nMore references can be added, which would help to expand different sections.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [] }, { "id": "83916", "date": "30 Apr 2021", "name": "Anand Marya", "expertise": [ "Reviewer Expertise Orthodontics", "dental diagnosis", "dentistry" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nFirst of all I would like to thank you for the editorial invitation and secondly I would like to complement the authors on their hard work and reading through it I found it to be a very well written review paper. Optical coherence tomography is slowly gaining popularity in the dental field and will surely gain more ground in future with an increased use. The authors have compiled the data associated with the uses of OCT well in this paper.\nMy suggestion would be to include the types of OCT and their uses along with a brief historical perspective that would add more value to this paper. Other than that it is a very well written paper and with few additions should be indexed.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-310
https://f1000research.com/articles/9-1256/v1	19 Oct 20	{ "type": "Study Protocol", "title": "A protocol to assess the risk of dementia among patients with coronary artery diseases using CAIDE score", "authors": [ "Fardina Rahman Omi", "Lingkan Barua", "Palash Chandra Banik", "Mithila Faruque", "Fardina Rahman Omi", "Palash Chandra Banik", "Mithila Faruque" ], "abstract": "Introduction: The impact of coronary artery disease (CAD) on the later development of dementia is not well studied globally. Therefore, this study aims to determine the long-term risk of dementia using a mobile application-based tool in addition to elucidating the contributing factors among CAD patients. Protocol: This ongoing cross-sectional study is collecting data from 285 stable CAD patients admitted to the “Ibrahim Cardiac Hospital and Research Institute” for coronary revascularization from August 2019 to July 2020. The patients were recruited using a convenient sampling technique due to economic and logistical issues. Data were collected through a face-to-face interview using a pretested semi-structured questionnaire. Physical parameters (blood pressure and anthropometry) were measured while maintaining the adequate privacy of the patients. The biochemical parameters analyzed by the hospital lab were also collected. The next phase of this study involves the use of a mobile application-based tool, “The Cardiovascular Risk Factors, Aging, and Incidence of Dementia (CAIDE)” risk score, to determine the risk factors associated with dementia. In addition, a descriptive statistical and inferential analysis will also be performed to determine the key contributing risk factors linked to the development of dementia. Ethics and dissemination: The study has been reviewed and approved by the Ethical Review Committee of Bangladesh University of Health Sciences. The results will be actively disseminated through peer-reviewed journals, conference presentations, social media, online news portal, the internet, and various community/stakeholder engagement activities. Conclusion: As a baseline study of the country, this study will fill a key knowledge gap in the pathway to the development of better interventions for dementia in Bangladesh. Outcomes from this study will also help with raising awareness on the association of mental health-related issues with cardiovascular diseases so that an improved cardiac rehabilitation program can be implemented in Bangladesh.", "keywords": [ "Risk of dementia", "coronary artery disease", "CAIDE score", "risk factors", "Bangladesh" ], "content": "Introduction\n\nDementia is one of the foremost reasons for disability and dependency among older people and affects approximately 50 million of the world's population. Among them, 60% are presently living in low-and middle-income countries1,2. Future projections indicate that the cumulative number of people with dementia will reach 82 million by 2030 and 152 by 20503. The prevalence of dementia among people over 65 years is roughly 5%, while 20–40% of the general population older than 85 years are affected by dementia4. Because of the demographic transition and decreased mortality rate, the number of older people is increasing worldwide and may exceed 1 billion by 20205. As dementia is a disease of the geriatric population, many of these older populations are at risk of developing dementia in the future. Therefore, immediate initiatives are required to be developed to prevent, treat, and rehabilitate them.\n\nRegion-wide distribution of incidence indicates that the burden of dementia is generally higher in Asian countries (~50%) compared to Europe (25%), America (18%), and Africa (8%)6. In the Asia Pacific region, the number of people with dementia will increase from 23 million in 2015 to almost 71 million by 20506. Bangladesh is an over-populated country (>160 million people) situated in the South-East Asia region where 12 million are over 60 years of age7. From 1974 to 2001, the number of elderly people in Bangladesh increased from 1.38 million to 7.59 million, with 37% within the age range of 60–64 and 63% over 658. Like other countries in Asia, it has the same prevalence of dementia both in urban and rural settings and the prevalence of questionable dementia is 11.5% and definite dementia is 3.6%9. However, these numbers are slightly lower than in other regions of the world9.\n\nAnnually, 50 million patients with dementia all around the world cost US$818 billion, indicating the allocation of 3% of the world’s gross domestic product (GDP) for the treatment and care of dementia patients2. However, it is difficult for a developing country like Bangladesh to allocate such an amount of GDP only for dementia care. Therefore, initiation of risk stratification and risk reduction strategies are of utmost importance to develop control measures to handle the current and upcoming surge of dementia in Bangladesh.\n\nAlthough dementia is thought to be a disease of those above 60 years, individuals who have a history of myocardial infarction or other vascular diseases (e.g. stroke, peripheral artery disease, and invasive procedures, including coronary bypass surgery or carotid endarterectomy) can be affected prematurely by this10. People with coronary artery diseases (CADs) have a higher chance of developing dementia because they have already been exposed to some kind of vascular damage10. Numerous vascular risk factors like systolic hypertension and diabetes mellitus are already known to be associated with cognitive impairments11. Systolic hypertension is an imperative modifiable risk factor for late-life cognitive impairment that can cause vascular dementia12. People with dementia often have difficulty describing the pain caused by angina and heart attacks13. Moreover, they often lose self-possession and develop a dependency on others to lead their everyday life14. As dementia, depression, and anxiety can happen simultaneously, it may aggravate social isolation and affect someone’s interest in doing physical exercise. Dementia can make it hard for a person to remember his/her medication schedule even if they have been doing it for years to mitigate the effects of different health conditions13.\n\nHistory of the illness, cognitive function testing with brain imaging, and blood testing are the key parameters that are commonly used for dementia identification and diagnosis. Screening the mass population for dementia diagnosis is not recommended and feasible15. Therefore, the development of a predictive tool for determining dementia risk that could allow health providers to take preventive measures is important16. According to the previous Cardiovascular Risk Factors, Aging, and Incidence of Dementia (CAIDE) study, patients with heart disease are more likely to develop dementia in their later stages of life17. However, this observation seems to be controversial and the relative importance of heart disease in the development of dementia may vary from population to population with different risk profiles17. In Bangladesh, there is a lack of data regarding dementia and thus the policymakers are unable to take an initiative to address this issue. Again, no dementia risk prediction tool has been applied and tested yet for the Bangladeshi population to generate baseline evidence for future research on this progressive disorder. As there is a lack of sufficient resources and healthcare access to the general population, the development of a cost-effective dementia risk assessment tool is extremely important. Therefore, this study aims to predict the risk of developing dementia in established CAD patients using a mobile application-based risk prediction tool; the CAIDE risk score. In addition to this, we are also planning to evaluate the factors that influence later life dementia among patients with established CAD.\n\n\nProtocol\n\nThis is an ongoing cross-sectional study planned from August 2019 to July 2020. Figure 1 shows the study procedure that started from the literature review through to the end of dissemination. Different study objectives are described below.\n\nHere month and duration-wise description of overall study procedure is illustrated.\n\nPrimary objective 1: Dementia risk prediction— we are categorizing the study subjects as per CAIDE risk score of dementia as low, mild, moderate, high and very high risk.\n\nPrimary objective 2: Burden related to dementia\n\n• Assess the burden of sociodemographic risk factors (age, sex, marital status, education level, occupation, economic background) among the study subjects\n\n• Evaluate the distribution of behavioral risk factors (tobacco use in the form of smoke and oral consumption, inadequate fruits and vegetable intake, physical inactivity) among the study subjects\n\n• Assess the personal and family history of chronic diseases (hypertension, diabetes, CAD, stroke, chronic kidney disease, dementia or Alzheimer's disease) among the study subjects\n\n• Determine the burden of anthropometric risk factors (overweight and obesity) among the CAD patients\n\n• Assess how these risk factors distribute in different risk categories of CAIDE score\n\n• Explore the biochemical and other diagnostic profile of the study population\n\nSecondary objective: Identify the influencing factors\n\n• Evaluate the association of cardiac biomarkers with the risk of dementia development\n\n• Identify the factors that influence the risk of dementia among CAD patients\n\n• Identify the relationship between the number of coronary vessels involved and the risk of developing dementia among the study subjects\n\nThe study is being conducted in Ibrahim Cardiac Hospital and Research Institute (ICHRI), a tertiary level cardiac hospital in Dhaka, Bangladesh. It is one of the eight affiliated institutes of the Diabetic Association of Bangladesh (BADAS) that has the objective to improve both preventive and curative cardiac care with quality services at an affordable cost. At the initial stage, it was conceived as an extension of the cardiac outdoor facility of another affiliated hospital, Bangladesh Institute of Research and Rehabilitation in Diabetes, Endocrine and Metabolic Disorders (BIRDEM), which is the World Health Organization Collaborating Centre on Diabetes, Endocrine and Metabolic Disorders, the only one of its kind in Asia. However, thereafter it was agreed that the project would best achieve its objectives from a stand-alone specialized cardiac hospital supported by the multidisciplinary hospital of BIRDEM for treatment of cardiac patients concurrently suffering from other medical conditions. It is a 150-bedded specialized cardiac hospital that has a cardiac emergency, inpatient, and outpatient services.\n\nOngoing data collection of this study involves patients with stable CAD who are admitted to ICHRI for revascularization at the inpatient department. A total of 285 patients (age range 40 to 59 years) are selected using a convenient sampling technique due to economic and logistical issues. As this is a Master’s thesis that has a pre-defined period, this non-probability sampling method will enable us to achieve the desired sample size in a relatively fast and cost-effective way. All the registered and eligible patients under the cardiac surgery department were invited to take part in the study. For this, the researchers counseled both the patient and their caregivers about the potential benefit of screening the future risk of dementia and the importance of their participation. The researchers also informed that the participation is voluntary and they have the right to withdraw themselves or refuse to answer any question. Before enrollment, patients are being screened as having “no cognitive impairment” based on the Bangla adaptation of mini-mental state examination (BAMMSE), a self-reported statement, and documented clinical history. The sample size is determined using the prevalence of vascular dementia among the Indian population (11.4%) extracted from published literature18, with a precision of 4% at a 95% confidence interval. The final sample size is adjusted considering 15% non-response rate. Thus, a total of 285 patients were selected for the study. Exclusion criteria include patients with a history of cognitive impairment, congenital heart diseases, clinically unstable, pregnant, and refusal to participate in the study.\n\nA semi-structured questionnaire is being used to collect data19. The questionnaire was prepared based on the study objectives and the conceptual framework (Figure 2). The qustionnaire has five components: (1) sociodemographic information (age, gender, marital status, educational status, occupation, monthly household income); (2) behavioral information (tobacco use-smoking and oral consumption, dietary servings of fruits and vegetables and work-related physical activity); (3) personal history of chronic diseases and medication (hypertension, CAD, diabetes, chronic kidney disease); (4) family history of chronic diseases (Alzheimer's disease, hypertension, CAD, stroke, chronic kidney diseases); (5) measurement (anthropometric - height, weight and body mass index [BMI]; blood pressure - systolic and diastolic; and investigation parameters - blood glucose, creatinine, troponin-I, HbA1C, lipid profile, angiogram, echocardiogram, and electrocardiogram). The behavioral part is comprised of relevant questions adapted from the STEPwise approach to Surveillance (STEPs) of Noncommunicable diseases risk factors questionnaire (version 3.2) of the World Health Organization (WHO) with appropriate modification20. The English questionnaire was translated into the Bengali language to maintain cultural sensitivity and understandability by the participants. Then the translated questionnaire was pre-tested to detect any inconsistency, unclear wording, or unusually long time taken to administer. To pre-test, ten men and ten women patients with stable CAD within the pre-selected age group were recruited randomly and interviewed by the trained data collectors (one-on-one, in-person, and face-to-face) in the cardiothoracic department of ICHRI. Before this, the objective and importance of pre-testing were explained to the participants and the invitation followed the similar approach mentioned in the previous section. To maintain consistency among the interviewers and the pretesting sessions, we used a pretest answer sheet to note verbal and nonverbal responses to the translated questionnaire. The collected responses were analyzed and interpreted based on the following parameters: trends in responses; fundamental flaws with the design or format; attractiveness; comprehension; acceptance; and relevance.\n\nIn this framework, interaction of different factors on the risk of dementia is illustrated. Here, risk of dementia is outcome variable and the factors are independent variables.\n\nCAD, cardiovascular disease; S. creatinine, serum creatinine; LVEF, left ventricular ejection fraction; HbA1c, haemoglobin A1C; FBG, fasting blood glucose; RBG, random blood glucose.\n\nThe data is collected by the research student with the help of a male and female assistant in the inpatient department of ICHRI. Data collection is taking place in three steps covering the five components of the questionnaire. In the first step, a face-to-face interview is organized to gather information on sociodemographic background, behavioral risk factors, and personal and family history of chronic diseases. In the second step, physical measurements are conducted with adequate privacy. Anthropometric measurements (height and weight) are carried out following a standard method described in “Noncommunicable disease risk factors survey Bangladesh 2010”21 and values are recorded in the checklist. Generalized obesity is determined by BMI according to the international guidelines of BMI22 and calculated as weight in kg/height in m2. Blood pressure is measured using an aneroid sphygmomanometer on the right arm in a sitting position with their hand in resting on the handle of the chair or some objects. Following resting for at least 15 minutes the first reading is taken, followed by a second reading after three minutes of resting interval. Systolic and diastolic measurements were taken in mmHg. The mean of the two measurements is used to determine the final value of blood pressure. In the third step, all the investigation (biochemical, angiogram, echocardiogram, and electrocardiogram) data is collected from the patient’s record file. The coronary angiogram report is used to determine the number of vessels involved in the disease process. The echocrdiogram report is used to assess the left ventricular ejection fraction.\n\nCAIDE risk score, a mobile application-based risk prediction tool, will be used to assess the long term risk (20 years later) of dementia using all the collected data. This is the first evidence-based tool to predict dementia risk in individuals in their late-life23. The tool uses the risk score model to predict the risk of developing dementia in the future among middle-aged and elderly people23. It was developed by the European Institute of Innovation and Technology (EIT) from a multi-mode project. The project partners were Karolinska Institute (Sweden), Research Institutes of Sweden (Sweden), Imperial College London (UK), PracSaitariSant Joan de Deu (Spain), and Erasmus University Medical Centre Rotterdam (the Netherlands). This tool has been externally validated to predict late-life dementia risk24 and was incorporated into the Finnish Geriatric Intervention Study to Prevent Cognitive Impairment and Disability (FINGER) trial. The CAIDE score application has also been found to motivate participants into mitigating their risk factors25.\n\nThe application includes a series of questions, concerning their lifestyle and medical condition, and then calculates a dementia risk score based on the answers. There are a total of 15 points against all of the variables and there is the flexibility of skipping cholesterol level and blood pressure measurements wherever appropriate. The tool then generates the percentage of risk and categorizes risk as low, mild, moderate, high risk, and very high.\n\nThe CAIDE risk score has never previously been applied within the Bangladeshi population and hence we will pre-test it in a sample of CAD patients before the final application. The variables of the tool are incorporated in the semi-structured questionnaire and pre-tested among the patients described earlier in the ‘Questionnaire development’ sub-section. When data collection will be completed, tool-specific information will be inputted and analyzed to detect any inconsistency. The research student will entry the necessary data using an android (version 7.0) mobile phone where the application (CAIDE risk tool) will be downloaded from the Google play store.\n\nStable CAD patients— defined as admitted patients with myocardial infarction or ischemic cardiomyopathy who are hemodynamically stable, free from acute symptoms, and ready for prescribed revascularization.\n\nCognitive impairment— defined according to BAMMSE in which any score < 24 points (out of 30) indicates cognitive impairment.\n\nCurrent tobacco user— those who have smoked or consumed tobacco orally in the past 30 days are considered to be a 'current' user21.\n\nInadequate fruit and vegetable intake— consumption of less than five servings of fruits and/or vegetables in a day is considered inadequate21.\n\nLow physical activity—we define it as per STEPS protocol and for that, we convert all the work-related physical activities in minutes per day (metabolic equivalent of task or MET-minute) as follows:\n\n• One minute in sedentary position (sitting quietly) = 1 MET-minute\n\n• One minute in moderate activities = 4 MET-minutes\n\n• One minute in vigorous activities = 8 MET-minutes\n\nThen all the MET-minutes will be added together to get the cumulative physical activity in MET-minutes. Based on the cumulative MET-minutes participants will be categorized as low, moderate, and high as follows: ≥3000 MET-minutes per week = highly active; 600–3000 MET-minutes per week = moderately active. The respondents who do not fall within the moderately active group will be categorized in the low activity group21.\n\nOverweight and obesity—we classify overweight and obesity as BMI 25.0 - 29.9 kg/m2 and BMI ≥ 30.0 kg/m2, respectively22.\n\nHypertension—it will be diagnosed based on “Seventh Report of Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure (JNC7)” criteria when systolic blood pressure ≥140 mmHg and/or diastolic blood pressure ≥90 mmHg or known case of hypertension or on antihypertensive drugs26.\n\nDiabetes mellitus—it will be diagnosed as fasting plasma glucose ≥7.0 mmol/l (126 mg/dl) or 2–h plasma glucose ≥11.1 mmol/L (200 mg/dl) or a known case of diabetes or on anti-diabetic drugs27.\n\nTo maintain quality, a quality control panel is formed by the researchers who are acting in different levels of study. The panel is comprised of a research student who is the principal investigator, three senior public health specialists with expertise in prevention and control of chronic diseases, and a cardiothoracic surgeon. The cardiothoracic surgeon and the student together assessed and will recruit stable CAD patients as per inclusion and exclusion criteria. The questionnaire will be filled, checked, and entered into the database by the student herself. The group of three public health specialists are monitoring the overall patient recruitment and data collection procedure. Besides them, an external monitoring panel of two researchers from other institutions was recruited to evaluate the overall procedures and comment on the performance. To prevent bias, the monitoring panel is being anonymized for the data collectors. The overall performance at the end of the study will be categorized as satisfactory based on three parameters: (1) mode of measurement; (2) maintenance of data collection environment; and (3) mode of administration of the questionnaire. In addition, to assure the quality of the study, we are also maintaining and following specific protocol: (1) pre-testing of the questionnaire and the tool; (2) use of the standard method of measurement as per the STEPS survey of Bangladesh 2010; (3) use of show cards for a better understanding of dietary servings and intensity of physical activities; (4) maintainance of adequate privacy during physical measurements; (5) ensuring the use of robust equipment for physical and biochemical measurements.\n\nData will be analyzed using the Statistical Package for Social Science (SPSS) version 20.0 for Windows (SPSS, Inc. Chicago. IL. USA). All estimates of precision will be presented at a 95% confidence interval (CI) in the tables. In this study, the p-value (two-sided) will be considered statistically significant at the threshold of p<0.05. Analyses are planned based on study objectives.\n\nDescriptive statistics. To address the primary objective 1 and primary objective 2, a descriptive analysis will be conducted using mean, standard deviation (SD), median with interquartile range (IQR), frequencies and percentages where appropriate.\n\nExploratory data analysis. To address the secondary objectives, an in-depth exploratory analysis will be applied. For normally distributed variables, the correlation between biochemical parameters and dementia risk percentages will be assessed using the Pearson correlation coefficient or the Spearman correlation otherwise. For continuous, normally distributed variables, we will use a t-test while comparing two variables and one-way analysis of variance (ANOVA) while comparing more than two variables. For continuous variables not normally distributed, we will use non-parametric tests: the Mann-Whitney U test to compare two groups, and the Kruskal-Wallis test to compare three or more groups.\n\nTo identify the relationship between the numbers of coronary vessels involved and the risk of dementia among the study subjects, we will employ the χ2 test or Fisher’s exact test, depending on the number of observations obtained in each considered category. Similar statistical analysis will also be applied initially to assess the association of different risk factors with the risk of developing dementia among CAD patients. Moreover, we will identify the influencing factors that are associated with increasing the long-term risk of dementia development using multivariate analysis. For this purpose, univariate analysis will be used to select variables according to the p-value obtained (p≤0.25) to include in the multivariate analyses28,29. For the multivariate analyses, we will run multinomial logistic regression considering “dementia risk categories” as the dependent variable and the “low risk” as reference. We will also calculate odds ratios (OR), and 95% CI for each independent variable. In the regression table, predictors with OR >1 will be presented. To ensure the presence of no multicollinearity, we will use the variance inflation factor (VIF) before the regression analysis. The STROBE (Strengthening the Reporting of Observational Studies in Epidemiology) guidelines will be used to report the results of our study.\n\nThe purpose of the study and data safety issues will be explained to the participants. All the participants will provide their informed written consent for inclusion in the study19. The study will be conducted following the Declaration of Helsinki, and the protocol has been approved by the Ethical Review Committee of Bangladesh University of Health Sciences (identification number: BUHS/BIO/EA/19/208). The permission of data collection has been provided by the appropriate authority of ICHRI. No personally identifiable information will be collected to maintain the anonymity of the participants. Patients have not been involved in the setting of the research question or outcome measures. They had no role in designing or implementing this work or interpretation of the results.\n\nThis study will bring no direct and immediate benefits to the patients. Indirect benefits include a contribution to understanding the underlying risk of dementia among patients with CAD. This will eventually increase the awareness around the importance of early lifestyle modification and the integration of mental health in the cardiac rehabilitation program. No important risk is foreseen as a direct result of the study.\n\nWe will apply a comprehensive strategy to disseminate the findings of the study. The strategy will use various communication platforms to reach out to a diverse range of beneficiaries: CAD patients, caregivers, cardiologists, psychiatrists, researchers, journalists, etc. We will use academic media (i.e., peer-reviewed journal articles, national and international conference presentations), social media (i.e., Facebook), online news portal, other platforms in the internet (i.e., links to study reports on the university website) and electronic mail (i.e., posting of study findings to participants and stakeholders) to disseminate the outcomes from this study. The study data will be made available publicly through an online repository using CC0 license.\n\nIt remains ongoing (permanently closed to additional enrollment but subjects continue to undergo research-related activities like checking for consistency, completeness, coding, analysis).\n\n\nConclusion\n\nStudies on dementia risk and its associated factors is scarce in Bangladesh. To the best of our knowledge, this is the first study that is addressing the long-term risk of dementia to determine different associated factors among people with CAD in Bangladesh. This study will act as a baseline to introduce a cognitive screening program to the local healthcare system. CAIDE scoring could be beneficial in the selection of high-risk patients for prompt and early intervention studies and for other uses of bespoke treatment. As it is a cross-sectional study, we would not be able to draw a definitive conclusion, however, this study is exploratory in nature. Further, large-scale studies are required to find out the effectiveness of the CAIDE risk tool. Clinicians can use the CAIDE risk tool to guide their assessments in terms of clinical consideration and cognitive screening after its validation in the Bangladesh context.\n\n\nData availability\n\nNo underlying data are associated with this article.\n\nZenodo: A protocol to assess the risk of dementia among patients with coronary artery diseases using CAIDE score. http://doi.org/10.5281/zenodo.403300619.\n\nThis project contains the following extended data within the file ‘Extended data file.pdf’:\n\n- Consent form (English & Bengali Version)\n\n- Interview questionnaire (English & Bengali Version)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nGBD 2015 Disease and Injury Incidence and Prevalence Collaborators: Global, regional, and national incidence, prevalence, and years lived with disability for 310 diseases and injuries, 1990-2015: a systematic analysis for the global burden of disease study 2015. Lancet. 2016; 388(10053): 1545–602. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLancet T: Dementia burden coming into focus. Lancet. 2017; 390(10113): 2606. PubMed Abstract \| Publisher Full Text\n\nWang W, Wu S, Cheng X, et al.: Prevalence of Alzheimer’s Disease and Other Dementing Disorders in an Urban Community of Beijing, China. Neuroepidemiology. 2000; 19(4): 194–200. PubMed Abstract \| Publisher Full Text\n\nJorm AF, Jolley D: The incidence of dementia: A meta-analysis. Neurology. 1998; 51(3): 728–33. PubMed Abstract \| Publisher Full Text\n\nDementia warning for the Asia-Pacific region. Lancet Neurol. 2015; 14(1): 1. PubMed Abstract \| Publisher Full Text\n\nPopulation ageing: a looming public health challenge. Health for the millions. 1998. (accessed 21 Jan2020). Reference Source\n\nBangladesh Bureau of Statistics (BBS): Population Census— 2001. Community Series. Bangladesh Bureau of Statistics, Ministry of Planning, Dhaka. 2003. Reference Source\n\nBarikdar A, Ahmed T, Lasker SP: The Situation of the Elderly in Bangladesh. Bangladesh Journal of Bioethics. 2016; 7(1): 27–36. Publisher Full Text\n\nPalmer K, Kabir ZN, Ahmed T, et al.: Prevalence of dementia and factors associated with dementia in rural Bangladesh: data from a cross-sectional, population-based study. Int Psychogeriatr. 2014; 26(11): 1905–15. PubMed Abstract \| Publisher Full Text\n\nPaciaroni M, Bogousslavsky J: Connecting Cardiovascular Disease and Dementia: Further Evidence. J Am Heart Assoc. 2013; 2(8): e000656. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDuron E, Hanon O: Vascular risk factors, cognitive decline, and dementia. Vasc Health Risk Manag. 2008; 4(2): 363–381. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSierra C: Hypertension and the Risk of Dementia. Front Cardiovasc Med. 2020; 7: 5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThe Dementia and Stroke or Coronary Heart Disease Toolkit - Stroke Foundation - Australia. - Stroke Foundation - Australia. Reference Source\n\nkoog I, Nilsson L, Palmertz B, et al.: A population-based study of dementia in 85-year-olds N Engl J Med. 1993; 328(3): 153–158. PubMed Abstract \| Publisher Full Text\n\nHarrawood A, Fowler NR, Perkins AJ, et al.: Acceptability and Results of Dementia Screening Among Older Adults in the United States. Curr Alzheimer Res. 2018; 15; 51–55. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHugo J, Ganguli M: Dementia and cognitive impairment: epidemiology, diagnosis, and treatment. Clin Geriatr Med. 2014; 30(3): 421–42. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRusanen M, Kivipelto M, Levälahti E, et al.: Heart diseases and long-term risk of dementia and Alzheimer's disease: a population-based CAIDE study. J Alzheimers Dis. 2014; 42(1): 183–91. PubMed Abstract \| Publisher Full Text\n\nShaji S, Bose S, Verghese A: Prevalence of dementia in an urban population in Kerala, India. Br J Psychiatry. 2005; 186: 136–40. PubMed Abstract \| Publisher Full Text\n\nRahman F, Barua L, Banik PC, et al.: A protocol to assess the risk of dementia among patients with coronary artery diseases using CAIDE score-Extended Data (Version 02) [Data set]. Zenodo. 2020. http://www.doi.org/10.5281/zenodo.4033006\n\nBonita R: Surveillance of risk factors for noncommunicable diseases: The WHO STEPwise approach: summary. Geneva: Noncommunicable Disease and Mental Health, World Health Organization. 2001. Reference Source\n\nNon-Communicable Disease Risk Factor Survey Bangladesh 2010. (Accessed 3, April 2019). Reference Source\n\nWorld Health Organization: Body mass index - BMI. World Health Organization. 2019. (Accessed, April 4, 2019). Reference Source\n\nKivipeltoM, Ngandu T, Laatikainen T, et al.: Risk score for the prediction of dementia risk in 20 years among middle aged people: a longitudinal, population-based study. Lancet Neurol. 2006; 5(9): 735–41. PubMed Abstract \| Publisher Full Text\n\nExalto LG, Quesenberry CP, Barnes D, et al.: Midlife risk score for the prediction of dementia four decades later. Alzheimers Dement. 2013; 10(5): 562–70. PubMed Abstract \| Publisher Full Text\n\nSindi S, Calov E, Fokkens J, et al.: The CAIDE Dementia Risk Score App: The development of an evidence-based mobile application to predict the risk of dementia. Alzheimers Dement (Amst). 2015; 1(3): 328–33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChobanian AV, Bakris GL, Black HR, et al.: The Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure: the JNC 7 report. Evidence-Based Eye Care. 2003; 4(3): 179–180. Publisher Full Text\n\nWHO: Definition and diagnosis of diabetes mellitus and intermediate hyperglycemia: report of a WHO/IDF consultation. 2006; 21. Reference Source\n\nBendel RB, Afifi AA: Comparison of Stopping Rules in Forward \"Stepwise\" Regression. J Am Stat Assoc. 1977; 72(357): 46–53. Publisher Full Text\n\nCostanza MC, Afifi AA: Comparison of Stopping Rules in Forward Stepwise Discriminant Analysis. J Am Stat Assoc. 1979; 74(368): 777–85. Publisher Full Text" }	[ { "id": "73348", "date": "14 Dec 2020", "name": "Shireen Sindi", "expertise": [ "Reviewer Expertise Epidemiology", "dementia", "lifestyle interventions", "dementia risk scores", "clinical trials", "stress", "sleep" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis protocol describes an exploratory study that aims to assess the risk of dementia among patients with coronary artery diseases (CAD) using the validated CAIDE score. The project will describe a sample of CAD patients and their CAIDE score. It will further measure different demographic, behavioral, and comorbidity risk factors for dementia. It will also assess the associations between various lab parameters with the risk score. This study is novel and unique for the Bangladeshi setting, and will better inform health care providers on the levels of dementia risk, including individual risk factors, which can support when planning dementia prevention initiatives. This project is also the basis for a Master’s thesis.\nI have the following questions/comments:\nINTRODUCTION\nIn the sentence “Systolic hypertension is an imperative modifiable risk factor for late-life cognitive impairment that can cause vascular dementia12.”. When describing dementia risk factors, I suggest that the authors use the terminology “increase the risk for” instead of “cause”.\n\nThe fourth paragraph can benefit from restructuring. It starts by describing vascular risk factors for dementia, but then switches to describing pain experienced by individuals who already have dementia. Then it moves on to describing depression, anxiety, isolation and lack of exercise. It ends with describing adherence to medication schedules. What is the goal of the paragraph? What idea is it trying to portray? What is the connection between these different components in this context? This would clarify the message to the reader.\n\nIs the observation of an association between heart disease and dementia really “controversial” as it is described? Even if it varies according to risk profiles? Please clarify or rephrase.\n\nSpecifically, which “mobile application-based risk prediction tool; the CAIDE risk score.” are the authors referring to? A search on App Store and Google Play does not show that such an App currently exists. If this App was used in the past, the exact name needs to be provided, as well as the dates when it was accessed/used.\nPROTOCOL\nStudy Design: How is this considered “an ongoing cross-sectional study”, when it was completed July 2020? Since it has been completed, can the authors present the study baseline data?\n\nPrimary objective 1: This is a description of what is done, not necessary an actual objective. Can this be rephrased into an objective?\n\nPrimary objective 2: What is meant by “biochemical and other diagnostic profile”: Please clarify.\n\nSecondary objective: Which cardiac biomarkers are you referring to?\n\nPlease provide more information regarding this objective: “relationship between the number of coronary vessels involved and the risk of developing dementia”.\nStudy population:\nThe text states: “As this is a Master’s thesis that has a pre-defined period, this non-probability sampling method…”. Can you please add more information about the pre-defined period?\n\nWas it a requirement to have a ‘caregiver’ to participate? Please clarify.\n\nRegarding verb tenses, if the study or a component of the study is complete, I suggest writing the descriptions in the past tense. For example it states “Before enrollment, patients are being screened as having”.\n\nIt is unclear what this statement is referring to “a self-reported statement, and documented clinical history”. Can you please clarify?\n\nWhy is the sample size based on vascular dementia prevalence, when this is a short-term cross-sectional study among middle-aged adults, with no measurements of dementia development?\n\nWhy is depression not included among the comorbid conditions?\n\nWhy does the behavioral information not include other dietary factors? E.g. fish consumption, consumption of meat, refined sugars?\n\nA more detailed description is needed for these parameters: “parameters: trends in responses; fundamental flaws with the design or format; attractiveness; comprehension; acceptance; and relevance.”\n\nThis statement is inaccurate “This tool has been externally validated to predict late-life dementia risk24 and was incorporated into the Finnish Geriatric Intervention Study to Prevent Cognitive Impairment and Disability (FINGER) trial.”. The EIT project (which took place later after the FINGER trial primary outcome results were published) included risk scores for late-life dementia. However, for the FINGER trial, the original midlife CAIDE Dementia risk score was used (not those for late-life)1.\nPLANNED ANALYSIS\nIt states “To identify the relationship between the numbers of coronary vessels involved and the risk of dementia among the study subjects, we will employ the X2 or Fisher’s exact test”. Why would X2 be used if both are continuous variables?\nETHICS\nWere some participants illiterate? If yes, how did they provide informed consent? Please clarify.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? No", "responses": [ { "c_id": "6277", "date": "25 Jan 2021", "name": "Lingkan Barua", "role": "Author Response", "response": "Thanks a lot for your valuable comment on our protocol. Here we have added our responses. Comments on Introduction Comment 1: In the sentence “Systolic hypertension is an imperative modifiable risk factor for late-life cognitive impairment that can cause vascular dementia12.” When describing dementia risk factors, I suggest that the authors use the terminology “increase the risk for” instead of “cause”. Response 1: Thank you for your valuable feedback, and we have changed it accordingly in the manuscript. Previously it was “Systolic hypertension is an imperative modifiable risk factor for late-life cognitive impairment that can cause vascular dementia” Now it is “Systolic hypertension is an imperative modifiable risk factor for late-life cognitive impairment that can increase the risk of vascular dementia” (Page number-3) Comment 2: The fourth paragraph can benefit from restructuring. It starts by describing vascular risk factors for dementia but then switches to describing pain experienced by individuals who already have dementia. Then it moves on to describing depression, anxiety, isolation, and lack of exercise. It ends by describing adherence to medication schedules. What is the goal of the paragraph? What idea is it trying to portray? What is the connection between these different components in this context? This would clarify the message to the reader Response 2: Good observation, and we have revised the manuscript as per the comments. However, here actually, we were trying to say if CAD patients somehow develop dementia, then what will be the after-effects. This is true that not all who will achieve risk score will develop dementia. But somehow if they develop then how negatively it will impact the overall situation of both disease conditions was the matter of focus. For making the concept reader-friendly, we have discarded the lines. However, we have discarded those lines as those lines are not that to the readers. (Page number-3) Comment 3: Is the observation of an association between heart disease and dementia really “controversial” as it is described? Even if it varies according to risk profiles? Please clarify or rephrase. Response 3: We have discarded these lines (page number-3) Comment 4: Specifically, which “mobile application-based risk prediction tool; the CAIDE risk score.” are the authors referring to? A search on App Store and Google Play does not show that such an App currently exists. If this App was used in the past, the exact name needs to be provided, as well as the dates when it was accessed/used. Response 4: Thank you for the valuable comment. We use the searchable tool named “Dementia Risk Tool” instead of “mobile application-based risk prediction tool; the CAIDE risk score.” as per your suggestion to find out quickly in the Google play store. We have attached the link reference (https://pubmed.ncbi.nlm.nih.gov/16914401/) of the mobile app tool for your kind reference. But after December this tool is not displayed at Google Play store anymore, but we have completed the assessment through this tool) (B). Comment on protocol Study Design: Comment 1: How is this considered “an ongoing cross-sectional study”, when it was completed in July 2020? Since it has been completed, can the authors present the study baseline data? Response 1: We have submitted the protocol in June 2020. Moreover, this protocol is based on an MPH thesis work, which we submitted in June 2020, and the duration was one year. Due to COVID Pandemic, all the thesis works were postponed due to different restrictions of the Government of Bangladesh. However, now again, we are working on it. Comment 2: Primary objective 1: This is a description of what is done, not necessarily an actual objective. Can this be rephrased into an objective? Response 2: We have assessed the level of future dementia risk and categorize them as low, moderate, and high using the CAIDE risk score Now it is “Primary objective 1: Dementia risk prediction— to assess the level of dementia risk of the study subjects as per CAIDE risk score and categorize as low, moderate and high” (Page number-4) Comment 3: Primary objective 2: What is meant by “biochemical and other diagnostic profile”: Please clarify Response 3: By biochemical profile, we meant the laboratory investigations of the respondents that were based on blood samples. We have evaluated the ECG findings, echocardiogram, and angiographic findings of the respondents, and as these are the tests that are a basis of a diagnosis of CAD patients, so we mentioned them as a diagnostic profile. However, we changed it to an investigation profile in the manuscript which would be more appropriate. Now the line is “Explore the biochemical and other investigation profiles (Echocardiogram, angiogram, electrocardiogram, etc. will also be included) of the study population” (Page number-5) Comment 4: Secondary objective: Which cardiac biomarkers are you referring to? Response 4: We are collecting the reports of Brain Natriuretic Peptide (BNP) and N-terminal prob-type Natriuretic Peptide (NT-pro-BNP) as a cardiac biomarker of the respondents which is our secondary objective. (Page number-5) Comment 5: Please provide more information regarding this objective: “relationship between the number of coronary vessels involved and the risk of developing dementia”. Response 5: We will explore the relationship between cardiac vessel involvements with dementia risk by applying multivariate analysis. Normally cardiac vessel involvement can be of single-vessel disease (SVD), Double vessel disease (DVD), and Triple vessel disease (TVD). We will find out if there is any variation in the risk category for vessel involvement. Now it is “Identify the relationship between the number of coronary vessels involved (Single vessel disease (SVD), double vessel disease (DVD) and triple vessel diseases (TVD)) and the level of dementia risk of the study subjects” (page number-5) Study Population Comment 6: The text states: “As this is a Master’s thesis that has a pre-defined period, this non-probability sampling method…” Can you please add more information about the predefined period? Response 6: Master’s thesis needs to be complete by a one-year duration, and in the case of this study, we have the period from July 2019 to August 2020. By this time, protocol development, pre-testing, data collections all need to be completed. However, due to the COVID 19 situation, our study is also compromised (university authority allows us six months more) like the rest of the world. Comment 7: Was it a requirement to have a ‘caregiver’ to participate? Please clarify. Response 7: No, it was not a requirement but based on the patients' disease severity; we sometimes took help from the caregivers. Comment 8: Regarding verb tenses, if the study or a component of the study is complete, I suggest writing the descriptions in the past tense. For example, it states, “Before enrollment, patients are being screened as having” Response 8: Thank you for the valuable suggestion, and we revised the manuscript accordingly (Page number-5) Comment 9: It is unclear what this statement is referring to as “a self-reported statement, and documented clinical history”. Can you please clarify? Response 9: We have gone through the participants' interviews (self-reported statement) and have gone through their clinical history, written by a certified physician (documented clinical history), with appropriate permission from the physician. Comment 10: Why is the sample size based on vascular dementia prevalence, when this is a short-term cross-sectional study among middle-aged adults, with no measurements of dementia development? Response 10: We consider this prevalence for the following reasons- a. There is a lack of dementia prevalence study at Bangladesh b. Available prevalence study is not representative to such group of population c. Our study population is more exposed to develop vascular dementia as they already have such risk factors d. We selected the urban area of our neighboring country as the food habit, lifestyles are almost similar. Comment 11: Why is depression not included among the comorbid conditions? Response 11: For the diagnosis of clinical depression, we need to use a few specific scales, which was not possible in those settings. Comment 12: Why does behavioral information not include other dietary factors? E.g. fish consumption, consumption of meat, refined sugars? Response 12: Study populations are from a very sensitive group, and they were getting prepared for bypass surgery, and many of them had restrictions of too much talking, movement. Taking all these into the ground, we tried to keep the questionnaire as short as possible. So, these dietary habits were not explored. However, we included the risk factors (included fruits and vegetable intake) of chronic diseases that are usually used by the STEPwise approach to Surveillance (STEPs) of the Noncommunicable diseases risk factors questionnaire (version 3.2) of the World Health Organization. Comment 13: A more detailed description is needed for these parameters: “parameters: trends in responses; fundamental flaws with the design or format; attractiveness; comprehension; acceptance; and relevance.” Response 13: By’ trends in responses,’ we mean whether the participants respond in the same pattern or do it vary from participant to participant? By ‘fundamental flaws with the design or format,’ we mean whether the data quality match with the study design or not? By ‘attractiveness,’ we mean, do the questionnaire materials attract the audience? By ‘comprehension,’ we mean, are the materials properly understood by the target participants? By ‘acceptance,’ we mean, is there anything in the questionnaire which is not sensitive or unfitting? By ‘relevance,’ we mean the facts we are talking about ever faced by the participants. We have elaborated them appropriately on page number-7 Comment 14: This statement is inaccurate “This tool has been externally validated to predict late-life dementia risk24 and was incorporated into the Finnish Geriatric Intervention Study to Prevent Cognitive Impairment and Disability (FINGER) trial.”. The EIT project (which took place later after the FINGER trial primary outcome results were published) included risk scores for late-life dementia. However, for the FINGER trial, the original midlife CAIDE Dementia risk score was used (not those for late-life Response 14: We have corrected those lines Now the line is “This tool has been externally validated to predict mid-life dementia risk 24 and was incorporated into the Finnish Geriatric Intervention Study to Prevent Cognitive Impairment and Disability (FINGER) trial” (C). Comment on PLANNED ANALYSIS Comment 1: It states “To identify the relationship between the numbers of coronary vessels involved and the risk of dementia among the study subjects, we will employ the X2 or Fisher’s exact test”. Why would X2 be used if both are continuous variables? Response 1: We will categorize the vessel involvement as SVD, DVD, and TVD and categorize the dementia risk as low, moderate, and high. After that, we will apply for X2. Now the line is “To identify the relationship between the numbers of coronary vessels involved (SVD, DVD, and TVD) and the level of dementia risk among the study subjects, we will employ the χ2 test or Fisher’s exact test” (Page Number-9) (D). Comment on Ethics Comment 1: Were some participants illiterate? If yes, how did they provide informed consent? Please clarify Response 1: We found none of the respondents as illiterate. Hence, we did not face any problem to take consent." } ] } ]	1	https://f1000research.com/articles/9-1256
https://f1000research.com/articles/8-55/v1	14 Jan 19	{ "type": "Case Report", "title": "Case Report: Rare presentation of adult intussusception at Orotta National Referral Hospital, Eritrea", "authors": [ "Senay Iyassu", "Million Abraha", "Senay Iyassu" ], "abstract": "A 38-year-old woman presented at Orotta National Referral Hospital emergency department in May 2017 with pain in the epigastric region and vomiting. Physical examination revealed no pertinent findings. Blood and urine tests were normal, and erect abdominal x-ray revealed a distended small intestine with multiple layers of “air-fluid levels”. CT scan and MRI were not done due to their temporary unavailability. During laparotomy a large mass of 20x20 cm in size was detected in the mid-jejunum of the small intestine. This leading tumor caused intussusception and coiling of the small intestine. As there are no typical symptoms of intussusception, it is very important to do CT scan for patients with long-standing abdominal pain and vomiting to achieve a definitive diagnosis of intussusception.", "keywords": [ "Intussusception", "Bowel obstruction", "Abdominal pain" ], "content": "Introduction\n\nIntussusception is commonly defined as telescoping of a proximal part of an intestinal loop towards the distal part of the loop1–6. The incidence of intussusception is 1–3 cases per 1,000,000 population per year2,7. The condition is more frequent in children than in adults1. Patients present sometimes with nonspecific symptoms8,9. The majority of patients come to hospital with abdominal pain and vomiting9. As these are unspecific symptoms, diagnosis of intussusception could be missed or even delayed before one would entertain intussusception as a cause of abdominal pain10. The unspecific presentation makes it difficult to define clearly the diagnosis before surgical operations1,11. Ordinarily, half of these cases are diagnosed in theater12. Helpful diagnostic tests include plain abdominal film13,14, abdominal CT scan15 and ultrasound16. Operative procedures to reduce the intestine is the treatment modality of intussusception1,17. In this paper we present a patient with intussusception with nonspecific symptoms who presented to Orotta National Referral Hospital, and emphasize the importance of early CT scan for quick and definitive diagnosis.\n\n\nCase report\n\nA 38-year-old female patient presented to Orotta National Referral Hospital, Asmara, Eritrea, in May 2017 with abdominal pain and vomiting of 3-month duration. The pain was diffuse, intermittent and more on the periumbilical area. She only vomited after ingesting food, otherwise she does not. There is no history of abdominal distension and she was passing gas. Her condition gradually worsened from time to time. There was no history of fever, no bloody vomiting, and no history of jaundice or urinary complaints. There was no known family history of intestinal obstruction.\n\nOn physical examination, the patient appeared sick and emaciated, although vital signs where within normal limits. She had slightly pale conjunctiva and dry mucosa. She had good air entry on both lungs with normal heart sound. There was diffuse tenderness on the abdomen but more on the epigastric region. There was no guarding or rebound tenderness. There was no any palpable mass on the abdomen until 1 day before the operation, when a large suprapubic mass was detected. Bowel sounds were present.\n\nLaboratory findings showed that complete blood count was within the normal limit. Hemoglobin of 10 g/dl (normal range, 12–16 g/dl), white blood cell count of 7×103/µl (normal range, 5–10 × 103/μl), platelet count of 350×103/µl (normal range, 150–400 × 103/μl). Urinalysis reveled no abnormal finding and blood chemistry values were aspartate transaminase levels of 57 U/l serum (normal range, 8–48 U/L), alanine aminotransferase level of 43 U/l (normal range, 7–55 U/l), amylase of 191 U/l (normal range, 23–85 U/l). Several plain abdominal x-rays were done with no pertinent findings. The last time plain abdominal x-ray was done showed a distended small intestine and with multiple “air-fluid levels”. Ultrasound of the abdomen depicted pockets of fluid collections. No abnormality was observed upon gastroscopic study (mild duodenal ulcer with excessive bile reflux). CT scan and MRI were not available.\n\nDespite the diagnostic challenges, several differential diagnoses were entertained, such as chronic peptic ulcer disease cholecystitis acute pancreatitis and partial intestinal obstruction and was managed conservatively. The patient was being treated as case of peptic ulcer disease before her surgical operation and was taking amoxicillin 1g twice daily and metronidazole 500mg twice daily for 10 days.\n\nAt 1 day before surgery, an abdominal mass was felt during repeated physical examination. Non-operative reduction using hydrostatic or pneumatic pressure by enema was not attempted. Surgical intervention was decided and performed 2 weeks after initial presentation, with an impression of small intestine obstruction secondary to malignancy. The patient was kept nothing per mouth (NPO) and was given normal saline intravenous fluid, broad-spectrum ceftriaxone (1 g intravenous twice a day) and metronidazole (500 mg intravenous three times a day). She was also given hydrocortisone (100 mg intravenous three times a day for 3 days). The patient was also administered 2 units of blood were before surgery. During laparotomy a large mass of 20×20 cm was identified in mid jejunum and a leading tumor had caused intussusception and coiling of the small intestine (Figure 1 and Figure 2). During laparotomy, intussusception reduction was attempted manually but the large intestine was too greatly looped, tangled and edematous to be reduced so resection was decided. Around 1 meter of the bowel was resected, and anastomosis was done in two layers. A biopsy of the leading tumor later showed it to be a simple hemangioma of the small intestine. In her postoperative management, she was kept NPO for 3 days and took intravenous fluids. Ceftriaxone and metronidazole were continued for 10 days. On her second day postoperatively, abdominal auscultation revealed the start of bowel sounds. She was discharged 2 weeks after surgery and was appointed to come for her follow-up after a month. In her follow up visit full remission was confirmed.\n\n\nDiscussion\n\nThe preoperative diagnosis of intussusception remains challenging as the patients usually have nonspecific symptoms during their presentation to medical facilities11. CT scans aid diagnosis considerably and usually shows a “bull’s-eye” sign18,19. Early in her presentation, the patient did not have signs of intestinal obstruction, but because of her persistent vomiting and epigastric pain other diagnoses were suggested. The diagnosis was greatly delayed partly because of diagnostic constraints. The temporary unavailability of the only CT scanner in the whole country made it difficult for early and accurate diagnosis. Looking at the importance of CT scan and MRI in the diagnosis of many medical and surgical conditions, it is highly recommended that the Eritrean Ministry of Health should import several additional CT scans or MRIs for a successful diagnosis and management of such health conditions. It is recommended as well to use CT scan as early as possible when a patient is suffering from a long-standing abdominal pain and vomiting.\n\n\nData availability\n\nNo data is associated with this article.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patient.", "appendix": "Grant information\n\nThe authors declare that no grants were involved in supported this work.\n\n\nReferences\n\nMarinis A, Yiallourou A, Samanides L, et al.: Intussusception of the bowel in adults: a review. World J Gastroenterol. 2009; 15(4): 407–411. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPotts J, Al Samaraee A, El-Hakeem A: Small bowel intussusception in adults. Ann R Coll Surg Engl. 2014; 96(1): 11–14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAref H, Nawawi A, Altaf A, et al.: Transient small bowel intussusception in an adult: case report with intraoperative video and literature review. BMC Surg. 2015; 15: 36. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShaheen K, Eisa N, Alraiyes AH, et al.: Telescoping intestine in an adult. Case Rep Med. 2013; 2013: 292961. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAnajar S, Tatari M, Hassnaoui J, et al.: [A rare case report of laryngeal leech infestation in a 70-year-old man]. Pan Afr Med J. 2017; 26: 19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDraiss G, Razzouki K, Mouaffak Y, et al.: Upper airway obstruction and hemoptysis due to a leech infestation in a child. Arch Pediatr. 2016; 23(1): 94–96. PubMed Abstract \| Publisher Full Text\n\nManouras A, Lagoudianakis EE, Dardamanis D, et al.: Lipoma induced jejunojejunal intussusception. World J Gastroenterol. 2007; 13(26): 3641–3644. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYakan S, Caliskan C, Makay O, et al.: Intussusception in adults: clinical characteristics, diagnosis and operative strategies. World J Gastroenterol. 2009; 15(16): 1985–1989. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYalamarthi S, Smith RC: Adult intussusception: case reports and review of literature. Postgrad Med J. 2005; 81(953): 174–177. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLianos G, Xeropotamos N, Bali C, et al.: Adult bowel intussusception: presentation, location, etiology, diagnosis and treatment. G Chir. 2013; 34(9–10): 280–283. PubMed Abstract \| Free Full Text\n\nMaghrebi H, Makni A, Rhaiem R, et al.: Adult intussusceptions: Clinical presentation, diagnosis and therapeutic management. Int J Surg Case Rep. 2017; 33: 163–166. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nReijnen HA, Joosten HJ, de Boer HH: Diagnosis and treatment of adult intussusception. Am J Surg. 1989; 158(1): 25–28. PubMed Abstract \| Publisher Full Text\n\nAydin N, Roth A, Misra S: Surgical versus conservative management of adult intussusception: Case series and review. Int J Surg Case Rep. 2016; 20: 142–146. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLe J, Labha J, Khazaeni B: The Malingering Intussusception. Clin Pract Cases Emerg Med. 2017; 1(4): 298–300. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEisen LK, Cunningham JD, Aufses AH Jr: Intussusception in adults: institutional review. J Am Coll Surg. 1999; 188(4): 390–395. PubMed Abstract \| Publisher Full Text\n\nRiera A, Hsiao AL, Langhan ML, et al.: Diagnosis of intussusception by physician novice sonographers in the emergency department. Ann Emerg Med. 2012; 60(3): 264–268. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKim KH, Namgung H, Park DG: Adult intussusceptions: preoperative predictive factors for malignant lead point. Ann Surg Treat Res. 2014; 86(5): 244–248. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMarsicovetere P, Ivatury SJ, White B, et al.: Intestinal Intussusception: Etiology, Diagnosis, and Treatment. Clin Colon Rectal Surg. 2017; 30(1): 30–39. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHarrison LE, Kim SH: Images in clinical medicine. Intussusception of the small bowel. N Engl J Med. 2004; 351(4): 379. PubMed Abstract \| Publisher Full Text" }	[ { "id": "58473", "date": "17 Feb 2020", "name": "Yifan Wang", "expertise": [ "Reviewer Expertise Gastrointestinal surgery" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nLyassu et al. describe a rare case of adult small bowel-small bowel intussusception caused by an intestinal hemangioma that was treated with surgical resection.\nThe diagnosis was challenging because of the lack of institutional availability of cross-sectional imaging (CT/MRI). It would be informative to add an image of the abdominal x-ray showing air-fluid levels. Also, it is unclear what the \"pockets of fluid collections\" visualized on abdominal ultrasound represent.\nIt would be interesting to show histopathological correlation of the intestinal hemangioma (H&E section). There are only a handful of such cases causing intussusception in the literature, and the authors may want to summarize this experience in the discussion.\nFigure 1. legend should read \"bowel\", rather than \"bowl\".\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "82133", "date": "13 Apr 2021", "name": "Gennaro Martines", "expertise": [ "Reviewer Expertise General surgery" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIntussusception is 1-5% of causes of intestinal ileus and double intussusception is an even more exceptional observation. CT and MRI are the gold standard for diagnosis but ultrasonography can be an effective diagnostic tool and demonstrates multiple concentric rings associated with an abdominal echogenic mass and echolucent rings representing the mucous covering of the intussuscepted bowel segment. I think that this description is due in the presentation of ultrasonography. for the differential diagnosis. Some English errors like bowl in figure 1. Add in references this article which well describe a case like this: Fabio Marino1 , Pierluigi Lobascio, Gennaro Martines, Angela D Di Franco, Marcella Rinaldi, Donato F Altomare Double jejunal intussusception in an adult with chronic subileus due to a giant lipoma: a case report Chir Ital Mar-Apr 2005;57(2):239-42.1\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-55
https://f1000research.com/articles/9-673/v1	03 Jul 20	{ "type": "Research Article", "title": "Factors influencing the utilization of dental services in East Java, Indonesia", "authors": [ "Ninuk Hariyani", "Dini Setyowati", "Multia Ranum Sari", "Diah Ayu Maharani", "Rahul Nair", "Kaushik Sengupta", "Dini Setyowati", "Multia Ranum Sari", "Diah Ayu Maharani", "Rahul Nair", "Kaushik Sengupta" ], "abstract": "Background: Despite high levels dental issues and insurance coverage in the East Java province Indonesia, the utilization of dental services is still low. This research aims to test some indicators for dental service utilization among East Java residents. Methods: A secondary analysis was undertaken using data on the East Java province from the Indonesian Basic Health Research 2013, which included 90,551 randomly selected respondents aged 5–100 years old. Socio-demographic characteristics (age, sex, education and residential location), dental behavior (tooth brushing habit), and clinical (dental) condition were self-reported through a questionnaire. Multivariable models were generated to estimate prevalence ratios (PR), and 95% confidence intervals (95%CI). Results: Prevalence of dental service utilization during the last 12 months in East Java province is only 9 %. Respondents 25–50 years old showed the highest utilization of dental services. Being male, having lower education and living in a district (as opposed to municipalities) were indicators for having lower utilization of dental treatment (PR [95% CI] = 0.81 [0.79–0.84], PR [95% CI] = 0.89 [0.86–0.93] and PR [95% CI] = 0.91 [0.88–0.95], respectively). Respondents with poor tooth brushing habit showed lower utilization of dental services. Having teeth was associated with higher utilization of dental treatment (PR [95% CI] = 1.39 [1.16–1.66). Conclusions: Age, sex, education and residential location influence the utilization of dental services among Indonesia’s East Java residents. Poor tooth brushing habits and being edentulous are also indicators of lesser utilization. These results call for urgent public health interventions to increase equitable dental care services utilization.", "keywords": [ "dental service utilization", "edentulism", "tooth brushing habits", "Indonesian", "population-based study" ], "content": "Introduction\n\nHealth is a fundamental right of every human being without discrimination related to race, religion, and socioeconomic status (World Health Organization, 2015). Oral health is integral to the overall health of human beings (Peres et al., 2019; World Health Organization, 2020). However, in most countries, access to and utilization of oral health services are limited (Glick et al., 2012; Petersen, 2003; Watt et al., 2019). Lack of access to such services can have a detrimental impact on people’s general health and quality of life (Petersen, 2003). Tooth loss is mainly the result of accumulated dental diseases as a product of low utilization of dental services (Petersen, 2003).\n\nOne of the commonly used indices to assess the utilization of dental services is the percentage of the population attending a dental visit in the previous year (Bayat et al., 2008). The utilization of dental services is varied across countries. In developing countries, the majority of people only visits the dentist for pain relief rather than preventive care (Varenne et al., 2006), while in developed countries about 40–80% of the adults visit a dentist in a given year (Bayat et al., 2008).\n\nPrevious research has indicated certain factors that influence dental service utilization. For example, socio-demographic factors related to dental service use include age, sex, education, and residential location. Moreover, poor health behaviors are usually clustered in the same person wherein a person with a bad tooth brushing habit also rarely accesses dental treatments (Jordao et al., 2018). Furthermore, dental clinical condition such as dental status (dentate vs edentulous) could influence the utilization of dental services as it differentiates the amount of dental treatment need. So far, utilization of dental services and factors related to it have been mainly reported in developed countries, while such reporting in developing countries has been limited.\n\nOral health is a much neglected field of research in developing countries, including in Indonesia. Indonesia is the fourth most populated country in the world after China, India, and the United States of America (United Nation, 2019). Cases of dental caries in this country is high; for example, more than 88% of the population has been estimated to have experienced caries, with 45% having untreated caries (The Ministry of Health, Republic of Indonesia, 2019). Currently, Indonesia is in the process of establishing universal health coverage through Jaminan Kesehatan Nasional (JKN), wherein basic dental health is included in the insurance coverage. This universal health coverage program was implemented as a capitation system whereby the dentists are paid a fixed amount for the number of people who were under their care (Deloitte Indonesia, 2019). The participants in the insurance scheme include Contribution Assistance Recipients (PBI) and non-PBI members (Deloitte Indonesia, 2019). PBI include poorer citizen whose insurance is funded by the government through taxes. Non-PBI members include other citizens not categorized as poor, who need to subscribe to the insurance scheme by paying for it monthly (e.g., through deduction directly from their income) (Deloitte Indonesia, 2019). A comprehensive assessment of the JKN program conducted by the Government of Indonesia in 2017 found that JKN has managed to bring 76% of Indonesia’s population under the program; this is considered an impressive coverage rate (Social Insurance Administration Organization, 2019).\n\nDespite the high level of dental problems and the high insurance coverage, the use of dental treatment among the Indonesian population is very low (The Ministry of Health Republic of Indonesia, 2013b). Indonesian Basic Health Research (Riset Kesehatan Dasar/RISKESDAS) 2018 showed that only 8.1% of Indonesians used dental services (The Ministry of Health Republic of Indonesia, 2013b). In Indonesia, East Java is the second most populated province with a slightly below average national dentist-population ratio (The Ministry of Health Republic of Indonesia, 2013a). The insurance coverage in this province is 80%. However, treat utilization for dental issues in this province is similar to the national estimate which is 8.6% vs 8.1%, respectively (The Ministry of Health, Republic of Indonesia, 2013b). Understanding the factors influencing dental service utilization among East Java residents is needed as a fundamental step to develop policy to increase the utilization of the services.\n\nTherefore, this study aimed to explore the associations of socio-demographic characteristics, behavioral factors, and clinical condition on the utilization of dental services among East Java residents.\n\n\nMethods\n\nThis secondary data analysis used data from the 2013 Indonesian Basic Health Survey (Riskesdas 2013). Riskesdas 2013 was a cross sectional national survey. It was part of a serial Indonesian national basic health survey conducted every six years. As the latest Riskesdas data is not currently open to the public, the Riskesdas 2013 data was used in this analysis. Riskesdas 2013 used a three-stage, stratified cluster sampling design to select a representative sample of Indonesian residents. The sampling frame was households recorded in the 2010 bloc census database, revalidated by the 2013 enumerator team. Indonesia was stratified into metropolitan and non-metropolitan areas by provincial status, with clusters based on district or municipality, which were selected with probability proportional to size. All persons in the household were included in the census. Final respondents were 294,959 households with the mean number of residents equal to 3.8. Response rate for the Indonesian residents was 93%. Details of the 2013 Indonesian national basic health research report has been published elsewhere (The Ministry of Health, Republic of Indonesia, 2013b). For the purpose of this analysis, a subset of East Java participants 5 to 100 years old who participated in the survey was analyzed.\n\nData was collected through a questionnaire in the Indonesian language. The outcome of interest was utilization of dental services. It was self-reported by respondents by answering a single question “Have you received dental treatment(s) during the last twelve months?” The response options were yes or no. Indicators of the dental service utilization were socio-demographic characteristics, behavioral factors, and clinical condition. Socio-demographic characteristics included age, sex, education and residential location. The inclusion criteria for the analysis were respondent aged 5–100 years old and completed the Riskesdas 2013 oral examination. Age was then categorized into ≤25 years old, 25–<50 years old, and ≥50 years old. The choice of the cut-off points for the age categorization was based in the distribution of the age. Sex was recorded as male or female. Education was measured by the highest level of school, post-school, or tertiary educational attainment and dichotomized into junior high school or less vs senior high school or higher. East Java province consists of 29 districts and nine municipalities, which differ due to the size of the area, capital, and development such as in the economy and education (where municipalities are usually ahead of the districts). Thus, residential location was dichotomized into districts and municipalities. The behavioral factor was tooth brushing habit (self-reported by the respondents as good vs bad tooth brushing habit). Respondents were categorized as having a good tooth brushing habit if they answered yes to the question “do you brush your teeth daily?”. Clinical condition was measured through dental status (self-reported by respondents as dentate (having one or more teeth) vs edentulous).\n\nStatistical analysis was performed using SAS version 9.4-callable SUDAAN version 11.0.3 (Research Triangle Institute, North Carolina, USA). Characteristics of the study participants were presented using descriptive statistics. Bivariate analyses of the association between utilization of dental treatment and each of the potential indicators were performed using chi square tests. The potential indicators include socio-demographic characteristics (age, sex, education and residential location), dental behavior (tooth brushing habit), and clinical (dental) condition. Multivariable logistic regression analysis was conducted to model together these factors influencing dental treatment utilization; no imputation was done for missing data. The statistical significance of the associations was evaluated at P < 0.05.\n\nEthical approval of Riskesdas 2013 was granted by the Ministry of Health Republic of Indonesia’s Human Research Ethics Committee. However, this particular study involved secondary analysis of anonymized data, and no new ethical clearance was required.\n\n\nResults\n\nA total of 90,551 respondents were included in this study. Study participants were aged between 5 and 100 years (mean: 36.9 [SD 20]) and comprised 48% males and 52% females. Table 1 shows the characteristics of the study participants. Only 23% of the respondents had education equal to or higher than senior high school, and only 19% of the study participants lived in municipality areas. Some 7.4% of the respondents reported having bad tooth brushing habit (rarely brushing their teeth) and 3% reported being edentulous. Only 9% of the respondents received dental treatment(s) during the last 12 months.\n\n95% CI: 95% Confidence Interval\n\nThe results from the bivariate analyses are presented in Table 2. All the indicators showed a significant relationship with the utilization of dental services. In terms of age, people over 50 years old showed the lowest utilization of dental treatment, followed by people less than 25 years old. Respondents that received the highest number of dental treatments were people between 25 and 50 years old. Furthermore, males, people with lower educational background, residents of districts, people reporting bad tooth brushing habit, and those with edentulism showed lower utilization of dental treatments compared to their counterparts.\n\nBold: indicator was significant; 95% CI: 95% Confidence Interval; PR: Prevalence Ratio; Bivariate analysis was conducted using chi-square\n\nThe results of the multivariable model are presented in Table 3. The model showed that dental services utilization differed by age. Respondents less than 25 years of age received lower dental treatment than respondents ≥50 years old (PR [95% CI] = 0.74 [0.71–0.78]). However, respondents 25–50 years old showed more utilization of dental treatments than respondents ≥50 years old (PR [95% CI] = 1.23 [1.18–1.28]). Being male and having lower education were indicators for having a lower utilization of dental treatment (PR [95% CI] = 0.81 [0.79–0.84] and PR [95% CI] = 0.89 [0.86–0.93], respectively). Similarly, people living in districts showed lower utilization of dental treatment than those living in municipalities. Respondents with bad tooth brushing habit showed lower utilization of dental treatment. Having teeth was associated higher utilization of dental treatment (PR [95% CI] = 1.39 [1.16–1.66]).\n\nBold: indicator was significant; 95% CI: 95% Confidence Interval; PR: Prevalence Ratio; Multi variable analysis was conducted using logistic regression\n\n\nDiscussion\n\nThis study showed that the utilization of dental services by East Java residents in Indonesia was very low. Only 9% of the East Java population received dental treatments during the last 12 months, which is slightly higher than the national average (8.1%) (The Ministry of Health, Republic of Indonesia, 2013b). Dental service utilization was low among the less than 25 years old respondents, male, district residents, edentulous people, and respondents with lower education and poor tooth brushing habit.\n\nAmong Indonesians, the fact that 95.5% of the population never utilize dental services was revealed in the 2018 national health survey (The Ministry of Health. Republic of Indonesia, 2019). The percentage of population that never access dental services in Indonesia was far higher than has been reported in India, another developing country (Gupta et al., 2014). Research in a rural population of western Rajasthan, revealed that around 55% of the population never visited a dentist while only 1.5% visited a dentist in the last 6 months (Gupta et al., 2014). However, these estimates contradict some research findings from economically developed countries where approximately 40%–80% of the population have accessed dental services within the last 12 months (Brown & Lazar, 1999; Kiyak & Reichmuth, 2005). Lack of awareness about oral health could be a reason behind the lower utilization of dental services in East Java province, Indonesia.\n\nIn East Java, utilization of dental services varied according to socio-demographic, behavioural, and clinical factors. People aged 25–50 years had the highest utilization of dental services in this study, followed by those ≥50 year old. Those less than 25 years old had the lowest utilization of dental treatment. Among Indonesians, people in 25–50 years of age are considered the productive workforce, at which point they have usually progressed in their careers to a point and are earning enough to allow them to have insurance or utilize dental services privately. This could be the reason for greater utilization of dental treatment in this age group.\n\nThis study finds that females have greater utilization of dental services, supporting previous studies (Emerich et al., 2015; Green & Pope, 1999; Honkala et al., 1997; Saintrain et al., 2014), while also contradicting another study (Akbar et al., 2019). Women’s greater use of oral health service providers is likely because they pay more attention to esthetics and oral hygiene. Research shows that women pay more attention to their appearance and health (Green & Pope, 1999). On the other hand, men tend not to seek dental service due to the lack of perception of their need (Kiyak, 1993).\n\nEducation is also an indicator for dental service utilization. People with lower educational background have lower access to dental treatment, supporting previous research. A study showed that higher educated individuals visited the dentist 10 times more than those with low education (Barros & Bertoldi, 2002).\n\nEast Java province is one of the provinces located in the main island, Java. Among other islands, the Java island is categorized as the most developed. Thus, socioeconomic inequality among each area in East Java could be considered lower than other less developed islands. However, this research still finds that dental service utilization is lower among the district residents than the municipality residents in East Java. This finding supports previous research (Akbar et al., 2019). It is known that public transport networks are less developed in rural areas than in urban areas. Lack of transport access to dental facilities can be an obstacle to routine dental visit (Hamano et al., 2017; Ogunbodede et al., 2015). Moreover, the use of a service also depends on the perception of user’s needs, influenced by their values, beliefs and cultures (Giordani et al., 2010). A study in Istanbul (Ozkan et al., 2011) found that more than half of the population surveyed did not feel the need or have the desire to visit a dentist, although their dental conditions were not ideal.\n\nThis study finds that people who have bad tooth brushing habit utilize dental services less, supporting previous research (Jordao et al., 2018). Jessor’s problem behaviour theory (Jessor, 1991) proposes that various risk behaviours are inter-related. Research (Jordao et al., 2018) has affirmed this theory in the dental behaviors field, finding that there are clustering patterns in dental health behaviors whereby less frequent tooth brushing is clustered with high sugar intake, current smoking, and lack of dental visits.\n\nMoreover, dentate people in East Java utilize more dental services than edentulous people, similar with other research finding (Tuominen, 1987). In many countries, the reason for dental service use is to mainly undergo dental treatment. This is especially true in developing countries where most of the people visit dental care services only when they are in pain (Varenne et al., 2006). Edentulousness in some parts of the world has been thought of as a healthy condition without pain even though this condition could reduce the ability to chew certain types of food, reducing the quality of life. Elders were also found to be more resilient to poor clinical status compared to younger people (Slade & Sanders, 2011).\n\nThe strength of this study lies in the nature of data collected in a national survey, allowing a representative data of the East Java population. The cross-sectional study design is a limitation as it precludes causal explanations. Self-reported utilization of dental services and lack of data on insurance coverage and income could be other limitations of this study as associations between lower socioeconomic status and decreased access to dental services have been found in several countries (Grytten et al., 2012; Hjern et al., 2001; Larson & Halfon, 2010; Listl, 2011; Murakami et al., 2014; Tchicaya & Lorentz, 2014). However, the study findings are in agreement with prior research that assessed dental care utilization, and it provides avenues for future research.\n\n\nConclusions\n\nThis first detailed population-based study in the East Java province of Indonesia has demonstrated that the use of dental services is influenced by socio-demographic factors such as age, sex, education and residential location. Moreover, poor tooth brushing habit and being edentulous are also indicators of lower dental service utilization.\n\n\nData availability\n\nData used for this analysis are available by a written request to the Ministry of Health Republic of Indonesia.\n\nThe available variables of Riskesdas 2013 dataset could be learnt from the national report of Riskesdas 2013 produced by Indonesian Ministry of Health, available online in http://labdata.litbang.kemkes.go.id/images/download/laporan/RKD/2013/Laporan_riskesdas_2013_final.pdf. A written request of a sub data set should be sent to the Ministry of Health Republic of Indonesia (sent to the head of research and development division of the Ministry of Health Republic of Indonesia at Jl. Percetakan Negara no 29 Jakarta Pusat, Indonesia) along with a proposal detailing the proposed analysis. After approval, the proposal will be analyzed by the data management laboratory. Successful applicants will get the data by email after signing a letter of agreement about the data management, including an agreement to neither send the data to other party nor using it for other reason than that has been agreed by the Ministry. The instruction of how to apply for the data are available from the Indonesian Ministry of Health’s website: http://labmandat.litbang.kemkes.go.id/images/download/peraturan/alur.pdf", "appendix": "References\n\nAkbar FH, Pasinringi S, Awang AH: Relationship Between Health Service Access to Dental Conditions in Urban and Rural Areas in Indonesia. Pesquisa Brasileira em Odontopediatria e Clínica Integrada. 2019; 19(1): e4652. Publisher Full Text\n\nBarros AJD, Bertoldi AD: Inequalities in utilization and access to dental services: a nationwide assessment. Ciência & Saúde Coletiva. 2002; 7(4): 709–17. Publisher Full Text\n\nBayat F, Vehkalahti MM, Zafarmand AH, et al.: Impact of insurance scheme on adults’ dental check-ups in a developing oral health care system. Eur J Dent. 2008; 2(1): 3–10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrown LJ, Lazar V: Dental care utilization: how saturated is the patient market?. J Am Dent Assoc. 1999; 130(4): 573–80. PubMed Abstract \| Publisher Full Text\n\nDeloitte Indonesia: Ensuring the Sustainability of JKN-KIS for the Indonesian People: Intended to cover the health costs of all Indonesians, the JKN-KIS national insurance program is now on the brink. How can this program survive?. Deloitte Indonesia Perspectives 1st ed. 2019. Reference Source\n\nDepartment of economic and social affairs - United Nation: World population prospect 2019: highlights. New York. 2019. Reference Source\n\nEmerich TB, Pacheco KTDS, Carvalho RB, et al.: Access to Dental Services and Related Factors in Adolescents from Vitória, Espírito Santo, Brazil, 2011. Brazilian Research in Pediatric Dentistry and Integrated Clinic. 2015; 15(1): 253–262. Publisher Full Text\n\nGiordani JMA, de Slavutzky SMB, Koltermann AP, et al.: Inequalities in prosthetic rehabilitation among elderly people: the importance of context. Community Community Dent Oral Epidemiol. 2010; 39(3): 230–8. PubMed Abstract \| Publisher Full Text\n\nGlick M, da Silva OM, Seeberger GK, et al.: FDI Vision 2020: Shaping the future of oral health. Int Dent J. 2012; 62(6): 278–291. PubMed Abstract \| Publisher Full Text\n\nGreen CA, Pope CR: Gender, psychosocial factors and the use of medical services: A longitudinal analysis. Soc Sci Med. 1999; 48(10): 1363–1372. PubMed Abstract \| Publisher Full Text\n\nGrytten J, Holst D, Skau I: Demand for and utilization of dental services according to household income in the adult population in Norway. Community Dent Oral Epidemiol. 2012; 40(4): 297–305. PubMed Abstract \| Publisher Full Text\n\nGupta S, Ranjan V, Rai S, et al.: Oral health services utilization among the rural population of western Rajasthan, India. J Indian Acad Oral Med Radiol. 2014; 26(4): 410–3. Publisher Full Text\n\nHamano T, Takeda M, Tominaga K, et al.: Is accessibility to dental care facilities in rural areas associated with number of teeth in elderly residents? Int J Environ Res Public Health. 2017; 14(3): 327. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHjern A, Grindefjord M, Sundberg H, et al.: Social inequality in oral health and use of dental care in Sweden. Community Dent Oral Epidemiol. 2001; 29(3): 167–174. PubMed Abstract \| Publisher Full Text\n\nHonkala E, Kuusela S, Rimpelä A, et al.: Dental Services Utilization Between 1977 and 1995 by Finnish Adolescents of Different Socioeconomic Levels. Community Dent Oral Epidemiol. 1997; 25(6): 385–90. PubMed Abstract \| Publisher Full Text\n\nJessor R: Risk behavior in adolescence: a psychosocial framework for understanding and action. J Adolesc Health. 1991; 12(8): 597–605. PubMed Abstract \| Publisher Full Text\n\nJordão LMR, Malta DC, Freire MdCM: Clustering patterns of oral and general health-risk behaviours in Brazilian adolescents: Findings from a national survey. Community Dent Oral Epidemiol. 2018; 46(2): 194–202. PubMed Abstract \| Publisher Full Text\n\nKiyak HA: Age and culture: influences on oral health behavior. Int Dent J. 1993; 43(1): 9–16. PubMed Abstract\n\nKiyak HA, Reichmuth M: Barriers to and enablers of older adults’ use of dental services. J Dent Educ. 2005; 69(9): 975–86. PubMed Abstract\n\nLarson K, Halfon N: Family income gradients in the health and health care access of US children. Matern Child Health J. 2010; 14(3): 332–342. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nListl S: Income-related inequalities in dental service utilization by Europeans aged 50+. J Dent Res. 2011; 90(6): 717–723. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMurakami K, Aida J, Ohkubo T, et al.: Income-related inequalities in preventive and curative dental care use among working-age Japanese adults in urban areas: A cross-sectional study. BMC Oral Health. 2014; 14: 117. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOgunbodede EO, Kida IA, Madjapa HS, et al.: Oral health inequalities between rural and urban populations of the African and Middle East region. Adv Dent Res. 2015; 27(1): 18–25. PubMed Abstract \| Publisher Full Text\n\nOzkan Y, Özcan M, Kulak Y, et al.: General health, dental status and perceived dental treatment needs of an elderly population in Istanbul. Gerodontology. 2011; 28(1): 28–36. PubMed Abstract \| Publisher Full Text\n\nPeres MA, Macpherson LM, Weyant RJ, et al.: Oral diseases: a global public health challenge. Lancet. 2019; 394(10194): 249–260. PubMed Abstract \| Publisher Full Text\n\nPetersen PE: The World Health Organization Report 2003: Continous improvement of oral health in the 21st century-the approach of the WHO Global Oral Health Programme. Geneva: World Health Organization. 2003. Reference Source\n\nSaintrain MV, Marques PL, Almeida LH, et al.: The relation between gender in the access to dental services and goods. Revista Brasileira em Promocao da Saude Fortaleza. 2014; 27(3): 381–388. Publisher Full Text\n\nSlade GD, Sanders AE: The Paradox of Better Subjective Oral Health in Older Age. J Dent Res. 2011; 90(11): 1279–85. PubMed Abstract \| Publisher Full Text\n\nSocial Insurance Administration Organization (Badan penyelenggara jaminan sosial kesehatan): Sampled data of Social Insurance Administration Organization sampel (Data BPJS kesehatan tahun 2015-2016). 2019; 1.\n\nTchicaya A, Lorentz N: Socioeconomic inequalities in the non-use of dental care in Europe. Int J Equity Health. 2014; 13: 7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThe Ministry of Health Republic of Indonesia. (n.d.): Health data and information of East Java Province: Executive summary (Ringkasan eksekutif: data dan informasi kesehatan propinsi Jawa Timur). Jakarta: Data and Information Center The Ministry of Health Republic of Indonesia. 2013a.\n\nThe Ministry of Health Republic of Indonesia: National report of Indonesian basic health survey 2018 (Laporan Nasional Riskesdas 2018). 2019. Tim riskesdas 2018. Lembaga Penerbit Badan Penelitian dan Pengembangan Kesehatan. Jakarta.\n\nThe Ministry of Health Republic of Indonesia: National report of Indonesian basic health survey 2013 (Laporan Nasional Riskesdas 2013). 2013b. Tim riskesdas 2013. Lembaga Penerbit Badan Penelitian dan Pengembangan Kesehatan. Jakarta.\n\nTuominen R: Utilization of oral health services by employed dentulous and edentulous populations. Proc Finn Dent Soc. 1987; 83(5–6): 249–255. PubMed Abstract\n\nVarenne B, Petersen PE, Fournet F, et al.: Illness-related behaviour and utilization of oral health services among adult city-dwellers in Burkina Faso: Evidence from a household survey. BMC Health Serv Res. 2006; 6: 164. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWatt RG, Daly B, Allison P, et al.: Ending the neglect of global oral health: time for radical action. Lancet. 2019; 394(10194): 261–72. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organization: Oral Health. 2020. Reference Source\n\nWorld Health Organization: Declaration of Alma-Ata. Am J Public Health. 2015; 105(6): 1094–5. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "66387", "date": "16 Jul 2020", "name": "Azlan Jaafar", "expertise": [ "Reviewer Expertise Dental Public Health" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article is scientifically-well-written. No major issues related to methods or data analysis need to be highlighted. The authors have indicated the problem statement in the introduction on why they conduct the study. However, comments were given to improve the flow and content of the section.\nThe method is well-describe. The analysis performed is matched with the study design as recommended by others. The outcome measure of dental service utilization is following a well-versed definition by other researchers. However, there are some categorisations of independent variables that do not match the results. For example the age and dental status. The author stated that they are using ≤25, 25-<50, and ≥50 years old. However, in the results it was mentioned differently (less than 25, 25-50, and ≥50. The age categorisation should not overlap thus I would like to suggest new categorisation <25, 25-50, and >50. Whereas, the dental status was informed in methods was “dentate vs edentulous” but in the results it mentioned, “not edentulous vs edentulous”. Although it seems to give similar meaning but consistency in reporting is essential.\nThe rest including abstract, discussion, and conclusion is well-written. The citation provided is also updated evidence. I also have given some papers for citations to enrich the discussion part. Other related information such as data sources is also stated in the identification of originality of the data.\n\nPlease see this annotated pdf.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6531", "date": "21 Apr 2021", "name": "Ninuk Hariyani", "role": "Author Response", "response": "Reviewer(s)' Comments to Author: Reviewer: 1. Azlan Jaafar Department of Periodontology & Community Oral Health, Faculty of Dentistry, Universiti Sains Islam, Nilai, Malaysia Comment 1:- The article is scientifically-well-written. No major issues related to methods or data analysis need to be highlighted. The authors have indicated the problem statement in the introduction on why they conduct the study. Response:- Thank you for your constructive feedback on our manuscript. Comment 2:- The method is well-describe. The analysis performed is matched with the study design as recommended by others. The outcome measure of dental service utilization is following a well-versed definition by other researchers. Response:- Thank you for your constructive feedback on our manuscript. Comment 3:- There are some categorisations of independent variables that do not match the results. The author stated that they are using ≤25, 25-<50, and ≥50 years old. However, in the results it was mentioned differently (less than 25, 25-50, and ≥50. The age categorisation should not overlap thus I would like to suggest new categorisation <25, 25-50, and >50. Response:- Thank you for the correction. The categorisations of < 25, 25-<50, and ≥50 has been used throughout the text except the one you pointed out. The change has been made to make it consistent throughout the manuscript. Comment 4:- The dental status informed in methods was “dentate vs edentulous” but in the results it mentioned, “not edentulous vs edentulous”. Although it seems to give similar meaning but consistency in reporting is essential. Response:- Thank you for the suggestion. The change of “dentate vs edentulous” has been made in Table 1, Table 2 and Table 3. Comment 5:- The rest including abstract, discussion, and conclusion is well-written. The citation provided is also updated evidence. Response:- Thank you for your constructive feedback on our manuscript." } ] }, { "id": "70071", "date": "11 Sep 2020", "name": "A. Gupta", "expertise": [ "Reviewer Expertise Public health", "oral health" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a very interesting paper but has some inconsistencies throughout. First, it is unclear whether the paper focusses on exploring factors influencing HIGH or LOW (or both) utilization of dental services in East Java, Indonesia. The background makes a case for LOW utilisation but the title and the overall conclusion state otherwise. Further, the discussion section is very superficial and will benefit from a thorough discussion of the study findings with the existing evidence. Some specific comments for the authors to consider are as follows:\n\nSpecific comments –\nTitle: Clarify whether the paper focuses on exploring factors influencing HIGH or LOW (or both) utilisation of dental services in East Java, Indonesia\nBackground:\n3rd paragraph: Support the sentences with more and relevant evidence.\n\n5th paragraph:\nIn the sentence, ‘Despite…..2013b)’ do you mean ‘use of dental treatment’ or ‘UTILIZATION OF DENTAL SERVICES’? Some clarity will be helpful.\n\nIn the sentence “However…..2013b)”, the phrase ‘treat utilization for dental issues’ needs clarity.\n\nSome discussion on the QUALITY of the public dental service will be helpful. Further, some statistics on ‘PRIVATE DENTAL CARE UTILIZATION’ as compared to the public dental service will be helpful.\n\nRevise the study aim for clarity- whether the paper aims to explore factors influencing HIGH or LOW (or both) utilization of dental services in East Java, Indonesia.\n\nMethods:\nPage 4, 2nd line: “Indicators of…..condition” seems inappropriate as it is meant to be the findings of this study. Suggest either deleting this sentence or adding the word ‘POTENTIAL’ at the start of this sentence. It will also be good if the authors can support this sentence with evidence, assuming that their choice of exposure variables was informed by previous literature.\n\nIn the same paragraph as above- in the sentence ‘East Java province….. districts), it will be helpful if the authors can expand on what they mean by ‘where municipalities are usually ahead of the districts’.\n\nStatistical Analysis: Authors mention ‘no imputation was done for missing data’ but no information (eg., n =?) on the missing data is provided. Please include the necessary information. Also, justification on how was missing data treated if no imputation was done will be helpful.\n\nResults:\nPlease insert the total sample size (n) in Table 1.\n\nDiscussion:\nOverall the discussion section in its current form is weak and superficial. It might be worth including a thorough and integrated discussion of the findings with the existing literature.\n\nPage 5: Sentences such as “Lack of awareness about oral health could be a reason behind the lower utilization of dental services” will be worth discussing a bit more.\n\nCan authors include a bit more discussion in each of the paragraphs below:\nIn the paragraph that talks about the difference in AGE for the utilisation- authors can include some discussion on Dental anxiety as one of the potential reasons for low utilisation among participants <25 years.\n\nIn the paragraph that talks about the GENDER difference for the utilisation- some more discussion on why there exists contradictory evidence will be helpful. Also supporting evidence stated is primarily from developed countries. Is there any evidence from developing countries that can support the justification- given that women's perspectives and beliefs may differ culturally?\n\nSuggest including similar discussions in other paragraphs on education, health behaviours, etc.\n\nIn the limitations section, it will be helpful if the authors can include some discussion on the potential of bias arising due to self-reported data and residual confounders.\n\nA paragraph on the research and policy implications of this study is missing and will be worth including. This study has the potential to inform guidelines, or specific changes to existing policy or practice in Indonesia.\n\nConclusion:\nA clear and precise conclusion will be helpful.\n\nPlease clearly state whether age (what age group?), sex (male/female), education (low/high), and residential location (district/municipal) are indicators of low utilization.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6532", "date": "21 Apr 2021", "name": "Ninuk Hariyani", "role": "Author Response", "response": "Reviewer: 2. A. Gupta University of Melbourne, Melbourne, Vic, Australia Comment 1:- This is a very interesting paper but has some inconsistencies throughout. Response:- Thank you for your constructive feedback on our manuscript. Point by point responses to your comments is provided. Comment 2:- Title: Clarify whether the paper focuses on exploring factors influencing HIGH or LOW (or both) utilisation of dental services in East Java, Indonesia Response:- The objective of this study was to see which of the included sociodemographic, behavioural, and clinical factors influenced the existing dental service utilisation. The study does not differentiate between low and high utilisation as the overall utilisation is low. To clarify this, we have detailed the study objectives in the abstract and the introduction. Comment 3:- Background: 3rd paragraph: Support the sentences with more and relevant evidence. Response:- Some references have been added to support and extend the topic. Comment 4:- Background: 5th paragraph: In the sentence, ‘Despite…..2013b)’ do you mean ‘use of dental treatment’ or ‘UTILIZATION OF DENTAL SERVICES’? Some clarity will be helpful. Response:- The sentence has been revised. Comment 5:- Background: In the sentence “However…..2013b)”, the phrase ‘treat utilization for dental issues’ needs clarity. Response:- Thank you for your comments. The consistent term of the utilization of dental services was used to reduce the ambiguity. Comment 6:- Background: Some discussion on the QUALITY of the public dental service will be helpful. Further, some statistics on ‘PRIVATE DENTAL CARE UTILIZATION’ as compared to the public dental service will be helpful. Response:- Some discussion has been added. Comment 7:- Background: Revise the study aim for clarity- whether the paper aims to explore factors influencing HIGH or LOW (or both) utilization of dental services in East Java, Indonesia. Response:- The aim of this study was to see which of the included sociodemographic, behavioural, and clinical factors influenced the existing dental service utilisation. The study does not differentiate between low and high utilisation as the overall utilisation is low. To clarify this, we has detailed the study objectives in the abstract and the introduction. Comment 8:- Methods: Page 4, 2nd line: “Indicators of…..condition” seems inappropriate as it is meant to be the findings of this study. Suggest either deleting this sentence or adding the word ‘POTENTIAL’ at the start of this sentence. It will also be good if the authors can support this sentence with evidence, assuming that their choice of exposure variables was informed by previous literature. Response:- Thank you for the suggestion. It has been adopted and the changes have been made. Comment 9:- Methods: In the same paragraph as above- in the sentence ‘East Java province….. districts), it will be helpful if the authors can expand on what they mean by ‘where municipalities are usually ahead of the districts’. Response:- Explanation has been added. Comment 10:- Methods: Statistical Analysis: Authors mention ‘no imputation was done for missing data’ but no information (eg., n =?) on the missing data is provided. Please include the necessary information. Also, justification on how was missing data treated if no imputation was done will be helpful. Response:- The total sample size of the final analysis has been added both in the method and Table 3 /multivariable analysis. The comparison of the characteristics of the total study participants and the final participants included in the multivariable analysis was also added in table 1 and the first paragraph of the results section. The included respondents were similar in all characteristics to the total number of respondents except in the educational level. The implication of this reduced sample size to the general finding has also has been added in the discussion. While previous article showed that people with lower educational background have lower access to dental treatment, this new article showed that due to the fact that the analysis sample in this study were better educated than the total respondent population, the influence of education on dental service utilization might only be underestimated. Comment 11:- Results: Please insert the total sample size (n) in Table 1. Response:- The sample size has been added as an additional column. We also provided a comparison of the characteristics of the total study participants and the final participants included in the multivariable analysis Comment 12:- Discussion: Overall the discussion section in its current form is weak and superficial. It might be worth including a thorough and integrated discussion of the findings with the existing literature. Response:- The discussion has been expanded in each of the suggested sections. Comment 13:- Discussion: Page 5: Sentences such as “Lack of awareness about oral health could be a reason behind the lower utilization of dental services” will be worth discussing a bit more. Response:- The discussion has been expanded. Comment 14:- Discussion: Can authors include a bit more discussion in each of the paragraphs below: In the paragraph that talks about the difference in AGE for the utilisation- authors can include some discussion on Dental anxiety as one of the potential reasons for low utilisation among participants <25 years. Response:- Discussion and literature on dental anxiety has been added. The discussion for each paragraph has been expanded. Comment 15:- Discussion: Can authors include a bit more discussion in each of the paragraphs below: In the paragraph that talks about the GENDER difference for the utilisation- some more discussion on why there exists contradictory evidence will be helpful. Also supporting evidence stated is primarily from developed countries. Is there any evidence from developing countries that can support the justification- given that women's perspectives and beliefs may differ culturally? Response:- The discussion has been expanded. Comment 16:- Discussion: Suggest including similar discussions in other paragraphs on education, health behaviours, etc. Response:- The discussion has been expanded. Comment 17:- Discussion: In the limitations section, it will be helpful if the authors can include some discussion on the potential of bias arising due to self-reported data and residual confounders. Response:- The discussion has been expanded. Comment 18:- Discussion: A paragraph on the research and policy implications of this study is missing and will be worth including. This study has the potential to inform guidelines, or specific changes to existing policy or practice in Indonesia. Response:- The discussion has been expanded. Comment 19:- Conclusion: A clear and precise conclusion will be helpful. Response:- The discussion has been expanded. Comment 20:- Conclusion: Please clearly state whether age (what age group?), sex (male/female), education (low/high), and residential location (district/municipal) are indicators of low utilization. Response:- The discussion has been expanded." } ] } ]	1	https://f1000research.com/articles/9-673
https://f1000research.com/articles/10-170/v1	02 Mar 21	{ "type": "Research Article", "title": "Public perceptions of the COVID-19 pandemic management in Bangladesh: a qualitative exploration", "authors": [ "Taufique Joarder", "Muhammad N.B. Khaled", "Mohammad A.I. Joarder", "Muhammad N.B. Khaled", "Mohammad A.I. Joarder" ], "abstract": "Background: Since the emergence of the COVID-19 outbreak, Government of Bangladesh (GoB) has taken various measures to restrict virus transmission and inform the people of the situation. However, the success of such measures largely depends on a positive public perception of the government’s ability to act decisively and the transparency of its communication. We explored public perceptions of pandemic management efforts by the Bangladeshi health sector decision-makers in this study. Methods: As this qualitative research was conducted during the COVID-19 pandemic, data was gathered through seven online mixed-gender focus group discussions involving 50 purposively selected clinicians and non-clinicians. Results: The study participants concurred that, from the outset, decision-makers failed to engage the right kind of experts, which resulted in poor pandemic management that included imposing lockdown in periphery areas without arranging patient transport to the center, declaring certain hospitals as COVID-19 dedicated without preparing the facilities or the staff, and engaging private hospitals in care without allowing them to test the patients for COVID-19 infection. Several participants also commented on ineffective actions on behalf of the GoB, such as imposing home quarantine instead of institutional, corruption, miscommunication, and inadequate private sector regulation. The perception of the people regarding service providers is that they lacked responsiveness in providing treatment, with some doctors misleading the public by sharing misinformation. Service providers, on the other hand, observed that decision-makers failed to provide them with proper training, personal protective equipment, and workplace security, which has resulted in a high number of deaths among medical staff. Conclusions: The Bangladeshi health sector decision-makers should learn from their mistakes to prevent further unnecessary loss of life and long-term economic downturn. They should adopt a science-based response to the COVID-19 pandemic in the short term while striving to develop a more resilient health system in the long run.", "keywords": [ "COVID-19", "Pandemic", "Resilience", "Health policy and systems research", "Bangladesh" ], "content": "Introduction\n\nIn December 2019, an unknown pneumonia-like disease appeared in Wuhan, China, but rapidly spread across the globe, prompting the World Health Organization (WHO) to label it as Coronavirus Disease 2019 (COVID-19)1. On 30 January 2020, the WHO declared a Public Health Emergency of International Concern (PHEIC) followed by pandemic declaration on 11 March 20202. In Bangladesh, the first case of COVID-19 infection was detected on 8 March, resulting in closure of educational institutions on 16 March. Following the first death on 18 March, in an attempt to contain the spread of the disease, the Government of Bangladesh (GoB) declared a ‘general holiday’ from 26 March to 4 April, which was repeatedly extended until 9 April, 14 April, 25 April, 5 May, 16 May, and 30 May. Despite these measures, COVID-19 infections continued to increase, but lockdown (which was formally termed by the government as ‘general holidays’) was withdrawn on 31 May3. The number of confirmed COVID-19 cases in Bangladesh exceeded 100,000 on 18 June, and the upward trend continued throughout summer, with 200,000 cases recorded on 18 July, 300,000 on 26 August, and 400,000 on 27 October4. By November 2020, Bangladesh ranked 24th and 20th in the world with respect to the total number of cases and deaths, respectively. At one point, Bangladesh ranked 3rd in terms of the number of new cases per day, but given that the country’s test rate (15,863 per million inhabitants) is among the lowest in the world (it is the second-lowest after Afghanistan among its South Asian neighbors) these figures are likely to be much higher5.\n\nPandemic response in Bangladesh is guided by the Infectious Diseases (Prevention, Control, and Elimination) Act 2018, which places the Directorate General of Health Services (DGHS) as the central coordinating and responsible body for COVID-19 response. Institution of Epidemiology, Disease Control, and Research (IEDCR) is the main scientific body to provide technical guidance and support for screening at the point of entry, and is in charge of imposing quarantine, managing contact tracing, and conducting initial testing (which was later contracted out to other government and a few private laboratories), while also providing forecasting and surveillance services, and the overall outbreak response6. This agency was highly criticized for monopolizing all the COVID-19 tests in the first three weeks following the detection of the first case in a country of 180 million inhabitants. It was also blamed by the medical community for severe shortages of personal protective equipment (PPE) and tests, due to which many health professionals refused to provide care to infected individuals7. To address this issue, from 3 April, approvals for additional test facilities were gradually issued, initially in the public and later in private facilities, totaling to 117 on 18 November8. However, even though GeneXpert equipment was already available for tuberculosis test, this antigen-based rapid test was only made available for COVID-19 test by the government in July 20209. Major events related to the COVID-19 pandemic in Bangladesh are shown in Figure 1.\n\nSince the COVID-19 outbreak, the GoB has taken various measures to inform the public of the situation and restrict the transmission of the infection. However, available evidence indicates that success of such measures largely depends on a positive public perception of government’s ability to manage pandemics effectively, as well as foster multi-stakeholder cooperation10, garner social order within the population10,11 and ensure good governance12,13. In this context, timely and transparent communication is essential14,15, as is involvement of technical and health experts in decision-making10,16. Gathering and sharing information on newly infected individuals and their contacts (as a part of contact tracing activities) is an important pandemic response activity, which can only be effective if the general public trusts the relevant agencies and service providers10,14,16. When people have a positive perception of the health system, they are more likely to adhere to any measures imposed to protect public health11,16,17. Thus, COVID-19 response requires adaptive leadership capable of making bold decisions and passing timely regulations based on the most recent scientific evidence, which is impossible without a positive perception of or trust on decision-makers and all pertinent stakeholders, including general public18.\n\nAlthough the epidemiologic features of SARS-COV-2 virus19,20, its clinical manifestations in different patient groups21,22, and its molecular characteristics23–25, as well as health systems response26, economic and social consequences27–29, and public attitudes toward the measures implemented in Bangladesh have been investigated30–32, public perceptions of pandemic management efforts by the responsible bodies have never been studied. Motivated by the work of Bigdeli et al., we decided to explore the public perceptions of COVID-19 pandemic management in Bangladesh by focusing on the relationships between (1) people and the decision-makers (or the larger health system governance), (2) people and the service providers (only physicians were covered in this study), and (3) service providers and decision-makers33. Findings yielded by this qualitative study will help decision-makers in introducing new or revising existing measures to allow service providers to better respond to the pandemic and increase public trust in the health system.\n\n\nMethods\n\nTo gather the data for this qualitative study, seven focus group discussions (FGDs) were conducted as a part of which participants’ perceptions of the COVID-19 pandemic management in Bangladesh were explored.\n\nAll the respondents provided verbal informed consent. All ethical principles were adhered to. The research was reviewed and approved by the Ethical Review Committee of the Public Health Foundation, Bangladesh (Reference number: 02/2020). During the pandemic, it was nearly impossible to gather all the participants of the FGDs and collect written consent. One may argue that it might be collected through digital signature. However, not all the participants were technologically well-equipped and trained. For convenience and treating all the participants’ consent in the same manner, we collected verbal consent. The Ethical Review Committee (ERC) approved this procedure.\n\nThe study was promoted via a Google Forms link circulated across social media and email databases. First, the Google Forms link was circulated on 19 May among the participants of a webinar on health system trust, organized by a youth organization, the United Nations Youth and Students Association of Bangladesh (UNYSAB). The link was further circulated across social media and email databases, requesting expression of interest to participate in the study. The email databases of the members of the Public Health Foundation Bangladesh and the UNYSAB have been used in this regard. For circulating the form, the link along with a request to fill-up the form, was posted in the network of social media groups of researchers, health professionals, and university-based organizations.\n\nThen, from the list of all the interested persons, the participants were purposively selected such that the FGD participants could be broadly classified into clinicians (graduate students with medical or dental background pursuing degrees in public health at a private university; renowned public health experts with a medical background; and clinicians practicing medicine or dentistry) and non-clinicians (undergraduate students pursuing non-medical degrees such as management, marketing, botany, business, and pharmacy, etc. at a public university; undergraduate students pursuing public health degrees at a public university; undergraduate students pursuing degrees in food and nutrition at a public university; and different professionals such as executives, trainers, managers, and coordinators of public and private organizations).\n\nPrior to commencing the FGDs, we developed a discussion guide in Bangla (see Extended data34), which was pilot tested in a session where a large group of health professionals and university students were present, organized by the UNYSAB, and adjusted in terms of issues addressed and the pattern of the language, where necessary. The goal was to focus discussions on the topics pertinent to this investigation, i.e., participants’ perceptions of COVID-19 pandemic management by the health sector decision-makers and the service providers, the implications of the actions (or lack thereof) taken by the decision-makers and service providers, and suggestions for improvements in pandemic management strategies.\n\nOnce a sufficient number of participants of both genders was recruited, seven FGDs (which were recorded through the video conferencing software Google Meet) were conducted between 15 and 17 June 2020, each involving 6−10 participants.\n\nThe FGDs were moderated by the first author (male), who has a doctorate in public health and is a health policy and systems researcher with experience and expertise in qualitative research methods. As a researcher in public health, he knew the participants in the FGD’s with the health professionals and these participants knew him as well. The second author (male), trained in economics with a Master’s degree and experienced in qualitative research, assisted in notetaking. He, however, had no prior engagement with any of the participants. Each FGD lasted 60−105 minutes and was conducted in Bangla, the native language of the respondents and the researchers. The research interests were explicitly explained to the participants, but the floor was open and no leading discussion points were initiated.\n\nPrior to content analysis—chosen due to the scarcity of existing literature on pandemic management in Bangladesh—the FGDs were transcribed by the research team35. For the diversity of the role among the FGD participants working in the health sector, data and observations were unique. In the FGD’s conducted among the university students, data saturation took place. Thematic analysis commenced with listening to the recordings and reading the transcripts, which allowed a coding schema to be developed in Microsoft Excel (version: Professional Plus 2016) based on the questions asked, noting the first impressions followed by labelling the text segments by newly emerging codes. Next, similar-meaning codes were merged and sorted into broader categories. To substantiate the emerging themes, appropriate excerpts from the FGDs were identified. In order to increase validity, the first and second author independently coded the dataset, seeking input from the third author in case of any disagreement.\n\n\nResults\n\nIn total, 50 individuals (28 males and 22 females, aged 19−75 years) took part in seven FGDs (Table 1). Four of these FGDs were held with individuals with a non-clinical background (n = 28) and the remaining three capture the views of clinicians (n = 22). Nearly half of the respondents had training in public health.\n\nParticipants’ perceptions regarding health systems decision-makers have been presented under three sub-themes: their perceptions regarding the preparatory phase of the pandemic (not appointing the appropriate professionals, leading to incorrect management steps), coordination (indecisions stemming from incoordination), and actions by the health sector decision-makers (miscommunication, poor regulation).\n\nPerceptions regarding preparation\n\nFor years, the Bangladeshi health system has been undermined by budget shortages, lack of quality services, high out-of-pocket payments, unregulated private sector, and a highly centralized secondary or tertiary care. These issues hindered the pandemic response, as the right persons were not placed in the right positions at the outset, as explained by a professor of public health:\n\n“An epidemic is a public health emergency; it is neither clinical nor an administrative issue. So, we must see this problem through the public health lens. We [epidemiologists] already know what to do to control an epidemic. … We the public health professionals should be given the flexibility that we are free to do whatever is needed for the country, not something that just pleases the political leadership.” [FGD-6, renowned public health experts, clinical background]\n\nFailure to engage the right professionals in the decision-making resulted in a rapidly escalating public health crisis, as the testing capacity, medical equipment and PPE provisions in the health centers were lacking. Study participants also concurred that the crisis was exacerbated by not instituting subsistence allowance for the poor before imposing lockdown (denoted by the GoB as ‘general holiday’), by failing to allow sufficient time for families to prepare for shop closures, and for increasing uncertainty by extending lockdown on a weekly basis. In addition, the health system actors wasted critical time by initially conducting tests at a single government facility before allowing private centers, although few in number, to engage in testing and provide COVID-19 care. Non-government organizations, and National Tuberculosis Control Program of the government had access to antigen-based rapid test, GeneXpert machines, which were utilized at a much later stage of the pandemic. As one student of public health explained:\n\n“Government is allowing, although lately, private diagnostic centers to perform COVID-19 tests. But there are many other machines which are not yet being utilized.” [FGD-2, graduate students of public health at a private university, clinical background]\n\nPerceptions regarding coordination\n\nSeveral FGD participants commented on lack of coordination which manifested through imposing lockdown in periphery areas of the country without arranging for the patients to be transported to the centrally located health facilities, declaring several hospitals as COVID-19 dedicated institutions without providing the required resources, and involving private hospitals in care provision without allowing them to test patients for COVID-19 on admission. Additionally, they were of view that lockdown was terminated due to the upcoming major Muslim religious holiday—the Eid—even though the outbreak was not showing any signs of abating. This decision was reached despite objections from the leading public health experts. Reflecting on these issues, a renowned public health expert remarked:\n\n“The civil administration must coordinate with the health system people at district and sub-district levels to ramp up the response against COVID-19. Success cannot be achieved without a tactful decentralization, involving different relevant ministries.” [FGD-6, renowned public health experts, clinical background]\n\nAccording to one FGD participant, lack of coordination contributed to the rapid spread of the disease among the low-income garment employees of Bangladesh:\n\n“BGMEA [national trade organization of garment manufacturers] ordered garment workers to return to Dhaka to save their jobs. After they returned by thousands, braving the coronavirus infection, BGMEA declared that the factories will not open. They [BGMEA] never consulted public health experts or health department. On the other hand, the police announced that they will not allow anyone to enter Dhaka. The innocent workers were caught between a rock and a hard place due to the lack of coordination between different departments.” [FGD-2, graduate students of public health at a private university, clinical background]\n\nPerceptions regarding actions taken by the health sector decision-makers\n\nSeveral actions by the health sector decision-makers were openly criticized by the study participants. They were particularly critical of the decision to impose home rather than institutional quarantine at the beginning of the pandemic, even though intimate Bangladeshi culture is not conducive to home quarantine. They were also of view that point-of-entry screening was weak, and blamed widespread corruption for PPE shortages, purchases of sub-standard equipment and mismanagement of relief materials. These issues, along with miscommunication, were frequently discussed in media. Several participants also criticized the decision to disguise lockdown as a ‘general holiday’ in order to reduce panic, while failing to allow the residents enough time to prepare. This was aptly surmised by one participant, who noted:\n\n“When they [government] say it is a ‘general holiday’ instead of ‘lockdown’, people confuse it with something festive. That’s why we saw people going to Cox’s Bazar [a popular tourist destination] for tourism purposes. Some of my friends even got married during this period, taking advantage of the ‘general holiday’.” [FGD-3, undergraduate students of public health at a public university, non-clinical background]\n\nPoor regulation was another complaint voiced by many participants, who were of view that it caused rapid escalation in prices of essential goods due to panic buying, while permitting uncontrolled advertisement and sales of unproven COVID-19 medicines (e.g., hydroxychloroquine, ivermectin, remdesivir, etc.). One participant commented on the proliferation of unauthorized and even fake testing centers that were providing false COVID-19-negative certifications:\n\n“Today, I saw in the news that someone who had been found corona negative here was found positive after landing in Japan. So, when things like these happen, our trust is compromised. These instances may even adversely affect our foreign relations.” [FGD-5, service holders of different professions, non-clinical background]\n\nParticipants’ perceptions regarding health service providers include lack of responsiveness of the providers, spreading misinformation, colluding in corruption.\n\nWhen the pandemic started, some newspapers alleged that, fearing for their own safety, some doctors were refusing to provide service to COVID-19 patents, or were not responsive enough while providing treatment. As one public university student explained:\n\n“In hospitals, especially the government hospitals, doctors don’t care about the patients. Doctors should not only provide clinical care, but also explain the disease, talk to the patient with respect, and provide more time.” [FGD-1, undergraduate students of different departments at a public university, non-clinical background]\n\nSome doctors were also accused of spreading misinformation through social and mainstream media. In a video that was rapidly disseminated across social media, one doctor confidently claimed that coronavirus would go away in the summer, while another respected senior doctor openly advertised unproven medicines on TV. These actions were condemned by a participant:\n\n“I found many of my doctor friends posting about different treatments for COVID-19. I think this may confuse and mislead people, as different doctors are saying different things.” [FGD-2, graduate students of public health at a private university, clinical background]\n\nSince the COVID-19 pandemic began, a doctor has been found colluding with someone guilty of running unauthorized testing centers36. Some were found promoting unproven medicines, and providing false certificates of COVID-19 negativity. One medicine (hydroxychloroquine) was initially included into the national treatment guidelines, only to be subsequently removed following the WHO’s warning about its ineffectiveness. A public health expert attributed these decisions to the vested interest of some clinicians serving on the technical committee in promoting certain treatments:\n\n“Some [doctors] are saying plasma therapy is the solution, some are promoting different other drugs like hydroxychloroquine even though the WHO is saying there is no specific treatment for COVID-19.” [FGD-6, renowned public health experts, clinical background]\n\nService providers’ perceptions regarding health systems decision-makers include leaving them unprepared and untrained in the face of the pandemic, not recognizing their sacrifices and the lack of workplace security stemming from COVID-19 mismanagement.\n\nDoctors that took part in this investigation felt that the health system decision-makers failed to prepare them adequately to combat COVID-19 effectively. They complained about lack of training, absence of treatment guidelines, PPE and equipment shortages, as well as inadequate food and logistic support while on duty. On this, a doctor that has been on COVID-19 duty since the pandemic outbreak said:\n\n“Doctors did not receive proper training or treatment guidelines, only online training, nothing on triage, how to handle indoor patients, no idea about treatment guideline, nothing on donning and doffing of PPE, or mental stress management. I feel like swimming in an unfathomable sea, without proper training.” [FGD-7, practicing clinicians, clinical background]\n\nThese issues resulted in a very high number of deaths among Bangladeshi doctors, which further undermined healthcare providers’ trust in the health system. Despite their sacrifices, they were not granted prioritized testing or healthcare, while also experiencing delays in salary payments. As a result, many doctors lacked motivation, as explained by one participant:\n\n“Government did not clarify direction regarding who would get the motivation package. Some doctors did not even receive their regular salary. This demoralized the doctors. … I know several young doctors who are saying that, if they are assigned COVID-19 duty, they will simply resign.” [FGD-6, renowned public health experts, clinical background]\n\nSeveral doctors expressed concerns over workplace security, as Bangladeshi people take out their dissatisfaction over the health system inadequacies on the doctors. To highlight the growing violence which resulted in a death of a colleague, many doctors stopped telemedicine services which they were previously offering benevolently to combat the COVID-19 crisis. A physician engaged in COVID-19 response said:\n\n“Decision-makers should make the work environment of the doctors safe in such a way that they themselves would confidently send their own children for treatment. Many of them [health sector decision-makers] have children who are doctors. I personally know several of them who are forbidding their children to serve in COVID-19 units, because there is no security there.” [FGD-7, practicing clinicians, clinical background]\n\n\nDiscussion\n\nThe findings yielded by this qualitative study indicate that several problems emerged as a consequence of failure to engage the right kind of experts in managing the pandemic. As a result of poor decision-making, the Bangladeshi health system was inadequately prepared to respond to the COVID-19 outbreak, as evident in the negative perception of our participants regarding the service providers especially in terms of quality of care they provided, and misinformation some of them shared in the social and mainstream media. Service providers also complained about lack of training, PPE, equipment, motivational packages, and workplace security.\n\nThe finding that the Bangladeshi health system failed to engage the right experts in the right positions is supported by several news articles and reports covering this topic. The Government of Bangladesh formed a 17-member National Technical Advisory Committee (NTAC) on 19 April 2020, more than a month after the first COVID-19 case was detected in the country. In the interim, most of the pandemic control efforts were entrusted to bureaucrats or administrators, many of whom lacked expertise or experience in health, let alone pandemic management. It is also worth noting that only three members of the NTAC had a public health career track37. This issue was further compounded on 21 April, when the government assigned 64 top bureaucrats to supervise and coordinate relief distribution activities in 64 districts of Bangladesh38 without seeking input or technical leadership from public health professionals. A policy analysis on the human resources for health in Bangladesh revealed that the DGHS is principally managed by the clinicians at the expense of public health experts. The same applies to the Ministry of Health and Family Welfare level, which comprises of members drawn from other ministries often unrelated to the health sector39. Given that such administrative approach is not conductive to pandemic management, lessons can be learned from Switzerland, Georgia, and New Zealand and other countries where science-based public health strategies have been proven highly effective40.\n\nSince doctors are often seen as the face of a health system, people blame them for any inadequacies in care delivery despite considerable sacrifices most doctors have made throughout the pandemic. So far, around 3,000 doctors in Bangladesh have contracted the virus and more than 100 have died due to COVID-1941. The negative perceptions regarding the service providers have been widely reported in Bangladeshi media42, which were attributed to poor communication skills and inadequate responsiveness (i.e., addressing the social needs of the patients such as being treated with friendliness, respect, information, trust, and sensitivity) in recent academic studies43–45.\n\nService providers’ claims that inadequate training, PPE and equipment shortages are the main cause of their grievances have also been documented in other studies from Bangladesh. In a study conducted from 9 to 14 April 2020, Islam and colleagues examined the frontline health workers’ perceptions and opinions on their personal safety while attending COVID-19 patients. Their findings show that 29% of the participating doctors lacked training on PPE use, 18% lacked training on COVID-19 case management, and 11% of the respondents did not receive any PPE46. Several news reports also highlighted the logistics issues related to food, lodging and transport provision for doctors working in COVID-19 dedicated hospitals47.\n\nWhen managing any health crisis, a science-based professional response is necessary and must involve relevant experts such as public health professionals, including infectious disease epidemiologists, health policy and systems experts, medical anthropologists, health economists, and health communication experts; laboratory scientists, including virologists, microbiologists, biochemists, and lab technicians; and relevant clinicians, including physicians, nurses, and paramedics. In the long run, however, a separate public health track, which is currently absent in the Bangladeshi health sector, must be implemented39. Service providers should be trained and directed to provide high-quality and efficient services with good quality and responsiveness48,49, while their legitimate demands should also be duly addressed.\n\nSince this study did not capture the perspectives of health decision-makers, it would be beneficial to conduct further investigations into health system governance incorporating their perspectives. Quantitative research should also be conducted to explore patients’ views on the responsiveness of the service providers, as well as service providers’ perspectives on their own safety and experiences during the COVID-19 pandemic.\n\nThe main limitation of this study stems from the use of online FGDs, which resulted in a sample that might not reflect the socioeconomic and demographic characteristics of the Bangladeshi population (as those without internet connectivity, or lower educational and socioeconomic status would be unable to respond to the Google Forms link or partake in online discussions). Consequently, the findings reported here cannot be generalized beyond the specific context in which the study was conducted. Second, it is worth noting that the first author was a COVID-19 patient at the time this study was conducted, which could potentially bias the qualitative analysis. However, every effort was made to reduce this risk through data triangulation50, and by engaging multiple research team members in data coding and interpretation.\n\n\nConclusions\n\nBangladesh experienced several local disease outbreaks over the past several years51–54 as well as a dengue epidemic in 201955, but due to their lower magnitude compared to the COVID-19 pandemic, the need for a comprehensive overhauling of the health systems has not been felt so deeply before. Low- and middle-income countries like Bangladesh are particularly vulnerable to pandemics due to their week governance and limited health system preparedness56. This article focused on the public perceptions of the pandemic management efforts by the health system actors, as the aim was to help the decision-makers and service providers in implementing more effective public health protection measures.\n\nThe main contribution of this investigation stems from highlighting the need to engage the right kind of experts in the right places at the outset of pandemic management efforts. It is further noted that public trust can be improved by being more transparent in official communications, while addressing the needs of service providers. These findings can help decision-makers revise their policies in order to prevent a longer-term loss of life and economic downturn. In addressing the COVID-19 pandemic or any future public health crisis, a science-based professional response is indispensable.\n\n\nData availability\n\nHarvard Dataverse: Extended data: “Public perceptions of the COVID-19 pandemic management in Bangladesh: a qualitative exploration”, https://doi.org/10.7910/DVN/CJZKJH34.\n\nThis project contains de-identified transcripts in Bangla.\n\nHarvard Dataverse: Extended data: “Public perceptions of the COVID-19 pandemic management in Bangladesh: a qualitative exploration”, https://doi.org/10.7910/DVN/CJZKJH34.\n\nThis project contains the following extended data:\n\n- Discussion guide used in the FDGs in both Bangla and English.\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgments\n\nWe thank the United Nations Youth and Students Association of Bangladesh (UNYSAB) for providing logistic support for data collection. 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BMC Res Notes. 2017; 10(1): 410. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRahman M, Hossain MJ, Sultana S, et al.: Date palm sap linked to Nipah virus outbreak in Bangladesh, 2008. Vector Borne Zoonotic Dis. 2012; 12(1): 65–72. PubMed Abstract \| Publisher Full Text\n\nSharmin S, Viennet E, Glass K, et al.: The emergence of dengue in Bangladesh: epidemiology, challenges and future disease risk. Trans R Soc Trop Med Hyg. 2015; 109(10): 619–627. PubMed Abstract \| Publisher Full Text\n\nMutsuddy P, Jhora ST, Shamsuzzaman AKM, et al.: Dengue Situation in Bangladesh: An Epidemiological Shift in terms of Morbidity and Mortality. Can J Infect Dis Med Microbiol. 2019; 2019: 3516284. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSriram V, Sheikh K, Soucat A: Addressing Governance Challenges and Capacities in Ministries of Health. S. Campbell, Editor. World Health Organization, UHC Partnership, Alliance for Health Policy and Systems Research: Geneva. 2020. Reference Source" }	[ { "id": "80588", "date": "24 Mar 2021", "name": "Theresa Hoke", "expertise": [ "Reviewer Expertise Public health", "implementation research", "health systems research." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors have conducted a timely study investigating an intriguing aspect of the COVID-19 pandemic: public perceptions of pandemic management efforts by Bangladeshi health sector leaders. They applied sound qualitative methods to answer the research question. Three types of relationships are considered: people and decision-makers; people and providers; and providers and decision-makers.\nThe authors are encouraged to consider the following points as they revise the paper to make it even stronger.\nSubstantive issues p.4 first column seems to indicate physicians were the only providers included, yet p.4 column 2 mentions clinicians practicing dentistry. In Bangladesh, are the latter considered physicians?\nIn reporting results, provide some indication of how prevalent the sentiment was across the focus groups. For example, was the issue mentioned in just one group, or in about half the groups, or in all the groups? Similarly, if there was a strong sentiment within a group, echoed by many participants, mention that. In reading the following text, for example, it was not obvious whether this was information derived directly from the FGDs, or if it was the authors’ perspective: “For years, the Bangladeshi health system has been undermined by budget shortages, lack of quality services, high out-of-pocket payments, unregulated private sector, and a highly centralized secondary or tertiary care.” On p.6, “Several FGD participants commented on lack of coordination….” Were these participants in a single group, or across several groups?\nOn p.6: “One participant commented on the proliferation of unauthorized and even fake testing centers….” Then on p.7: “Since the COVID-19 pandemic began, a doctor has been found colluding with someone guilty of running unauthorized testing centers”. Can these matters be reported together?\np.7: “Service providers’ perceptions regarding health systems decision-makers include leaving them unprepared and untrained in the face of the pandemic, not recognizing their sacrifices and the lack of workplace security stemming from COVID-19 mismanagement.” This text reads like a summary of findings rather than results coming directly from the FGDs. Consider moving that text to the Discussion.\np.7 The authors are encouraged to carefully consider how news articles are used to support research findings. How much confidence can be placed in the fundamental accuracy of news articles? Additionally, is it possible that news articles support what study participants say because study participants have been influenced by what they heard in the press?\nLimitations: Undergraduate students represent a major portion of the non-clinician participants. This is a legitimate approach, but this particular group does bring a special perspective, since university students tend to be younger and of higher socio-economic status than the population at large. Any potential bias could be mentioned in the Limitations section.\nMinor issues\nCheck wording in the following places:\nFigure 1: March 26—Scores of people. June 16: Resumption of commercial…\nReplace FGD’s with FGDs in a couple of places.\nTake another look at this wording: p.5 “For the diversity of the role among the FGD participants working in the health sector, data and observations were unique.” The meaning is not completely clear.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6503", "date": "29 Mar 2021", "name": "Taufique Joarder", "role": "Author Response", "response": "We have found the comments to be very thoughtful. We will address the comments and revise our manuscript soon, along with a point-by-point response. We thank the reviewer for taking the time to review and allow us to improve our manuscript." }, { "c_id": "6564", "date": "20 Apr 2021", "name": "Taufique Joarder", "role": "Author Response", "response": "p.4 first column seems to indicate physicians were the only providers included, yet p.4 column 2 mentions clinicians practicing dentistry. In Bangladesh, are the latter considered physicians? Response: In Bangladesh, MBBS (Bachelor of Medicine and Bachelor of Surgery) and BDS (Bachelor of Dental Surgery) are considered equivalent degrees, and both the holders of MBBS and BDS degrees are treated similarly at the professional level. For example, job circulars in the public sector specify MBBS and BDS degrees for applicants while referring to them as 'doctors' in general. The regulatory authority for providing medical practice license in Bangladesh, the Bangladesh Medical and Dental Council (BMDC), recognizes MBBS and BDS degree-holders as 'doctors,' and restrict others (for example the Sub-Assistant Community Medical Officers, village doctors, etc.) from using a 'Dr.' before their name to indicate their clinical qualification. In reporting results, provide some indication of how prevalent the sentiment was across the focus groups. For example, was the issue mentioned in just one group, or in about half the groups, or in all the groups? Similarly, if there was a strong sentiment within a group, echoed by many participants, mention that. In reading the following text, for example, it was not obvious whether this was information derived directly from the FGDs, or if it was the authors’ perspective: “For years, the Bangladeshi health system has been undermined by budget shortages, lack of quality services, high out-of-pocket payments, unregulated private sector, and a highly centralized secondary or tertiary care.” On p.6, “Several FGD participants commented on lack of coordination….” Were these participants in a single group, or across several groups? Response: This is a very useful suggestion. We have now provided some indication of how prevalent the sentiment was across the FGDs. On p.6: \"One participant commented on the proliferation of unauthorized and even fake testing centers….\" Then on p.7: \"Since the COVID-19 pandemic began, a doctor has been found colluding with someone guilty of running unauthorized testing centers\". Can these matters be reported together? Response: Sure. We have now removed the statement from p.7 to p.6 to present them together. p.7: \"Service providers' perceptions regarding health systems decision-makers include leaving them unprepared and untrained in the face of the pandemic, not recognizing their sacrifices and the lack of workplace security stemming from COVID-19 mismanagement.\" This text reads like a summary of findings rather than results coming directly from the FGDs. Consider moving that text to the Discussion. Response: Based on the initial review comment by the F1000Research reviewer, we added a summary of findings at the beginning of each sub-section. The part indicated in the comment is, in fact, the summary of findings. If deemed unacceptable by the reviewer, we have to discuss this issue again with the editorial team and resolve it. p.7 The authors are encouraged to carefully consider how news articles are used to support research findings. How much confidence can be placed in the fundamental accuracy of news articles? Additionally, is it possible that news articles support what study participants say because study participants have been influenced by what they heard in the press? Response: Various authentic media outlets in Bangladesh widely circulated the news articles reported in this manuscript. Given the nature of the issues reported, we think that the news articles substantiate the respondents' expressed views. We agree that there is a possibility of the participants being influenced by the press. However, this issue, in our opinion, will not invalidate our findings because the study's objective was to explore the public perceptions of COVID-19 pandemic management in Bangladesh, and many factors, including the media, naturally influence public perceptions. Limitations: Undergraduate students represent a major portion of the non-clinician participants. This is a legitimate approach, but this particular group does bring a special perspective, since university students tend to be younger and of higher socio-economic status than the population at large. Any potential bias could be mentioned in the Limitations section. Response: Agreed. We have now added this issue in the Limitations, as suggested. Check wording in the following places: Figure 1: March 26—Scores of people. June 16: Resumption of commercial… Replace FGD's with FGDs in a couple of places. Response: Thanks for identifying these errors. We have revised the text. Take another look at this wording: p.5 \"For the diversity of the role among the FGD participants working in the health sector, data and observations were unique.\" The meaning is not completely clear. Response: We revised the sentence as follows: \"For the diversity of the role among the FGD participants working in the health sector, data saturation was not achieved.\" We hope the meaning is clear now." } ] }, { "id": "80584", "date": "26 Mar 2021", "name": "Manuela Villar Uribe", "expertise": [ "Reviewer Expertise Public health", "Primary Health Care", "Measurement of Quality of Care", "Health Policy" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for inviting me to review this insightful manuscript. It comes at a very important time when discussions on lessons learned will help strengthen the health system in the short and medium term. The article is well written and presents results clearly and adequately. I only have three comments that could help strengthen the manuscript:\n\nThe participants in the focus groups are primarily students, health workers or health system experts. I suggest the results and title of the manuscript are framed more clearly to ensure the reader understands that the views presented are not actually representative of the general public but are rather the views of the health community in Bangladesh. Presenting and discussing the findings in light of the representation that the participants provide of a public health and medical population is also extremely valuable and better targeted for use in system strengthening activities to come: trust and coordination with and among medical professionals probably ought to have been a key component to the COVID response.\n\nI am not sure I understand why you have two sections in the results where you outline issues related to decision-makers. I suggest creating a joint section for those two sub-sections where you can highlight the role they played and allow the reader to foresee areas of improvement at the decision-maker level.\n\nI am particularly interested by policy recommendations and would encourage the authors to strengthen that section further by outlining recommendations in line with the results that are presented: what recommendations stem from the views on coordination, preparation, decision-makers, etc?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "6504", "date": "29 Mar 2021", "name": "Taufique Joarder", "role": "Author Response", "response": "We have found the comments to be very thoughtful. We will address the comments and revise our manuscript soon, along with a point-by-point response. We thank the reviewer for taking the time to review and allow us to improve our manuscript." }, { "c_id": "6565", "date": "20 Apr 2021", "name": "Taufique Joarder", "role": "Author Response", "response": "The participants in the focus groups are primarily students, health workers or health system experts. I suggest the results and title of the manuscript are framed more clearly to ensure the reader understands that the views presented are not actually representative of the general public but are rather the views of the health community in Bangladesh. Presenting and discussing the findings in light of the representation that the participants provide of a public health and medical population is also extremely valuable and better targeted for use in system strengthening activities to come: trust and coordination with and among medical professionals probably ought to have been a key component to the COVID response. Response: Thanks for the suggestion regarding the title of the manuscript. Since not all the participants belong to the health community, we edited the title a bit differently, which now reads as follows: \"Urban educated groups' perceptions of the COVID-19 pandemic management in Bangladesh: a qualitative exploration.\" We have now also edited the results such that the readers can understand whose views were presented and how prevalent the sentiment was across the FGDs. I am not sure I understand why you have two sections in the results where you outline issues related to decision-makers. I suggest creating a joint section for those two sub-sections where you can highlight the role they played and allow the reader to foresee areas of improvement at the decision-maker level. Response: This study was motivated by the work of Bigdeli et al. (2020)[1]. In this article, the authors proposed the Health Systems Governance Framework, adapted from the World Development Report 2004. This framework discusses relationships between three different spheres: 1) Between the Policymakers and Providers, 2) Between Providers and the People, and 3) Between the People and the Policy-Makers. We mentioned this in the last paragraph of the Background section. We explored the public perceptions of COVID-19 pandemic management in Bangladesh by focusing on the relationships between (1) people and the decision-makers (or the larger health system governance), (2) people, and the service providers (only physicians were covered in this study), and (3) service providers and decision-makers. Aligned with the objective of the article, the three sub-sections under the Result section have been organized. Creating the joint section will undermine the alignment with the research objectives. [1] Bigdeli M, Rouffy B, Lane BD, Schmets G, Soucat A. Health systems governance: the missing links. BMJ Global Health. 2020 Aug 1;5(8):e002533. I am particularly interested by policy recommendations and would encourage the authors to strengthen that section further by outlining recommendations in line with the results that are presented: what recommendations stem from the views on coordination, preparation, decision-makers, etc? Response: Thank you for this suggestion. While reviewing the manuscript, we clearly saw the weaknesses in the Recommendations section. We have now revised the entire section according to the advice of the reviewer." } ] } ]	1	https://f1000research.com/articles/10-170
https://f1000research.com/articles/9-1241/v1	15 Oct 20	{ "type": "Brief Report", "title": "Post-COVID recovery: characteristics of chronically critically ill patients admitted to a long-term acute care hospital", "authors": [ "Meg Stearn Hassenpflug", "Dale Jun", "David R. Nelson", "Tamas Dolinay", "Dale Jun", "David R. Nelson", "Tamas Dolinay" ], "abstract": "Background: Survivors of COVID-19 pneumonia often suffer from chronic critical illness (CCI) and require long-term hospitalization. Long-term acute care (LTAC) hospitals are vital in the care of CCI patients, but their role for patients post COVID-19 infection is not known. Barlow Respiratory Hospital (BRH) is a 105-bed, LTAC hospital network serving ventilator-dependent and medically-complex patients transferred from the ICUs of hospitals in southern California. We report patient characteristics of our first series of COVID-19 survivors admitted to the post-acute venue of an LTAC hospital. Methods: Single-center observational descriptive report of patients recovering from acute infectious complications of COVID-19 pneumonia requiring long-term respiratory support. Results: From 28 April to 7 September 2020, 41 patients were admitted to BRH for continued recovery from COVID-19 pneumonia. The length of stay at the transferring hospital was twice that of non-COVID patients admitted during the same time period. Median age: 68 [44-94] years, 61% male, 80.5% with tracheostomy, 51.2% on invasive mechanical ventilation, 22% receiving hemodialysis. All mechanical ventilation and hemodialysis interventions were initiated at the transferring hospital. Conclusions: To our knowledge, this is the first report to characterize CCI and medically complex COVID-19 patients transferred to the post-acute venue of an LTAC hospital. Patients on average spent over six weeks in the transferring hospital mostly in the ICU, are largely elderly, carry the known risk factors for COVID-19 infection, and experienced respiratory failure necessitating prolonged mechanical ventilation via tracheostomy. Our findings suggest that these patients will continue to require considerable medical interventions and treatments, including weaning from mechanical ventilation, owing to the numerous sequelae of the infection and the burden of acute-on-chronic diseases. As ICU survival rates improve, this research further emphasizes the important role of the LTAC hospital in responding to the COVID-19 crisis.", "keywords": [ "COVID-19", "post-acute", "chronic critical illness", "long-term acute care", "mechanical ventilation", "tracheostomy", "recovery" ], "content": "Introduction\n\nAdvances in technology, research, and adoption of evidence-based practices have significantly improved intensive care unit (ICU) survivorship, creating the population of patients recognized as chronically critically ill (CCI)1. This improved survival, however, is often accompanied by a prolonged and challenging course of recovery. This population now includes ICU survivors of coronavirus disease 2019 (COVID-19) in need of post-acute care for continued recovery from their infection. Long-term acute care (LTAC) hospitals are vital in the care of CCI patients2, but their role for patients post COVID-19 infection is not known. Barlow Respiratory Hospital (BRH) is a 105-bed, not for profit, LTAC hospital network serving ventilator-dependent and medically complex patients transferred from the ICUs of hospitals in southern California. Herein, we report patient characteristics of our first series of COVID-19 survivors admitted to the post-acute venue of an LTAC, as an essential step in the continuum of care for treatment, rehabilitation, and recovery.\n\n\nMethods\n\nThis is a single-center observational descriptive report of patients recovering from acute infectious complications of COVID-19 pneumonia requiring long-term respiratory support. Over half were admitted on invasive mechanical ventilation having experienced respiratory failure at the transferring hospital. Patients were admitted for attempts at weaning from prolonged mechanical ventilation, as well as for continued care and treatment of infections, complications, and co-morbid conditions.\n\nThe study was approved by the Western Institutional Review Board (WIRB), reference: #1-1348082-1. Only de-identified health information was collected and recorded in the database to ensure patient privacy and data safety. The WIRB waived the need for consent from patients who participated in the study.\n\nPatients with at least one positive COVID-19 polymerase chain reaction testing (PCR) prior to admission to BRH were enrolled in the study on an ongoing basis. The tests were performed from nasopharyngeal, oropharyngeal or lower airway sampling. Exclusion criteria of the study was the absence of positive COVID-19 PCR testing prior to admission. This approach was followed to minimize biases in data collection. Due to the inherent false negative rate of the PCR testing, it is possible that we did not capture all previously COVID-19 positive patients3. Patients were determined to be in the post-infective phase prior to transfer to BRH.\n\nThe data are reported with binary values. The 0 represents absence and 1 represents presence of a condition (see Underlying data). Missing data are reported as unknown. Data were collected from our electronic medical record system using a combination of automated data extraction and manual collection. We collected baseline demographics (age, gender, race/ethnicity, premorbid location), presence of known COVID-19 risk factors, events at transferring hospital, and descriptors of status on admission to the LTAC to construct the Barlow COVID-19 data set.\n\nAll statistical analysis was performed using Microsoft Excel 2013 program (Microsoft Corporation, Santa Rosa, CA). We used descriptive statistics to describe the basic features of the data. Missing data were omitted from analysis. For the variables, serum albumin and serum glucose, n=36 and n=40 respectively. No statistical comparisons were made.\n\nWe used the STROBE cross sectional reporting guidelines to report this research4.\n\n\nResults\n\nOf 194 patients transferred to BRH from 28 April 2020 to 7 September 2020, 41 (21%) were admitted for continued recovery from confirmed COVID-19 pneumonia. Selected demographics and patient characteristics are shown in Table 1. The length of stay at the transferring hospital was twice that of non-COVID patients admitted during the same time period. All mechanical ventilation and hemodialysis interventions were initiated at the transferring hospital. Table 2 presents treatment interventions already in effect on admission to BRH, descriptive characteristics, and laboratory values.\n\n\nDiscussion and conclusions\n\nLTAC hospitals provide specialized care for patients suffering from CCI5. With increased survival in the ICU, the number of patients transferred to these hospitals has also increased in the past decades2. Early reports of the COVID-19 pandemic indicate that 5-12% of patients with COVID-19 infection require ICU hospitalization6–8. These numbers suggest that the role of LTAC hospitals will expand during the COVID-19 pandemic, due in part to their ability to treat patients with illnesses and conditions that do not follow a linear trajectory of improvement.\n\nTo our knowledge, this is the first report to characterize CCI and medically complex COVID-19 patients transferred to the post-acute venue of an LTAC hospital. Patients on average spent over six weeks in the transferring hospital mostly in the ICU, are largely elderly, carry the known risk factors for COVID-19 infection, and experienced respiratory failure necessitating prolonged mechanical ventilation via tracheostomy. Patients present with physiological imbalances, numerous penetrating and indwelling catheters and disruptions of skin integrity breaching host defenses, and manifestations of allostatic load burden.\n\nOverall, our findings suggest that these patients will continue to require considerable medical interventions and treatments, including weaning from mechanical ventilation, owing to the numerous sequelae of the infection and the burden of acute-on-chronic diseases. As ICU survival rates improve, this research further emphasizes the important role of the LTAC in responding to the COVID-19 crisis. LTAC hospitals will play an increasingly critical function to fill gaps in our preparedness and response to COVID-19 infection by resuming and relieving care initiated in the acute hospital setting.\n\nOur analysis is limited by several factors: it is a single center descriptive report, with a small cohort of patients, and a still emerging evidence base for COVID and post-COVID infection. Patient characteristics from this single center study may not be applicable to other centers or the post-COVID pneumonia population in general due to geographic differences in patient demographics, referral patterns, and facility-specific treatment capabilities. Efforts to quantify disease burden and report the number and variety of interventions may be warranted to objectify the intensity of treatment at the LTAC hospital. In addition to reporting broad outcomes (wean rate, length of stay, discharge disposition) a goal is to identify subgroups of patients and craft specific clinical outcomes.\n\n\nData availability\n\nOpen Science Framework: Database, https://doi.org/10.17605/OSF.IO/VHJZG9. Registered 8th October 2020 (https://osf.io/2c8q9).\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nNelson JE, Cox CE, Hope AA, et al.: Chronic critical illness. Am J Respir Crit Care Med. 2010; 182(4): 446–454. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKahn JM, Benson NM, Appleby D, et al.: Long-term acute care hospital utilization after critical illness. JAMA. 2010; 303(22): 2253–2259. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWoloshin S, Patel N, Kesselheim AS: False Negative Tests for SARS-CoV-2 Infection - Challenges and Implications. N Engl J Med. 2020; 383(6): e38. PubMed Abstract \| Publisher Full Text\n\nvon Elm E, Altman DG, Egger M, et al.: The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) Statement: guidelines for reporting observational studies J Clin Epidemiol. 2008; 61(4): 344–9. PubMed Abstract \| Publisher Full Text\n\nKahn JM, Werner RM, David G, et al.: Effectiveness of long-term acute care hospitalization in elderly patients with chronic critical illness. Med Care. 2013; 51(1): 4–10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGrasselli G, Pesenti A, Cecconi M: Critical Care Utilization for the COVID-19 Outbreak in Lombardy, Italy: Early Experience and Forecast During an Emergency Response. JAMA. 2020; 323(16): 1545–1545. PubMed Abstract \| Publisher Full Text\n\nYoung BE, Ong SWX, Kalimuddin S, et al.: Epidemiologic Features and Clinical Course of Patients Infected With SARS-CoV-2 in Singapore. JAMA. 2020; 323(15): 1488–1494. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGuan WJ, Ni ZY, Hu Y, et al.: Clinical Characteristics of Coronavirus Disease 2019 in China. N Engl J Med. 2020; 382(18): 1708–1720. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDolinay T, Jun, D, Hassenpflug MS, et al.: Database. 2020. http://www.doi.org/10.17605/OSF.IO/VHJZG" }	[ { "id": "77597", "date": "08 Feb 2021", "name": "Anuj Mehta", "expertise": [ "Reviewer Expertise Critical care outcomes", "shared decision-making", "prolonged mechanical ventilation" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for allowing me the opportunity to review “Post-COVID recovery: characteristics of chronically critically ill patients admitted to a long-term acute care hospital” for consideration for your journal. The authors present an early case series of patients in the post-COVID-19 state who were admitted to an LTAC. They describe a significant burden of disease that highlights the need for ongoing care after initial recovery from acute COVID-19 illness. The authors present the experience of Barlow Respiratory Hospital which is a unique and large LTAC with a large referral base. The primary strength of the manuscript is that it presents novel data and gives a picture of the ongoing burden of disease related to COVID-19. However, the two major gaps are a sense of outcomes from the LTACH (which may be difficult to quantify as some patients may still be receiving treatment) and a sense of context with comparisons to the prior year. I have listed my specific concerns below. The source data is not included but is presented in an appropriate manner.\n\nMajor Comments\nThe question that naturally stems from the authors description is what happened to these patients. It would be helpful for the authors to present some outcomes data including percent successfully weaned, percent with ongoing ventilation, percent died, percent transferred back to a short term acute care hospital, percent discharged home etc. It would also be helpful to understand patients’ functional status if the data is available. Such descriptions would be helpful to truly quantify not only the burden of disease but the likelihood of recovery.\n\nThe authors’ data indicates that 51.2% of post COVID-19 patients required MV and 22% required RRT of some form. It is well known that during the first spike in COVID in the spring, there was a substantial drop in other disease states presenting to the hospital. In essence, for a while most hospitals were seeing COVID and nothing but COVID. I am unclear as to how much this was true in Southern California but it is a phenomenon seen in multiple parts of the country. Therefore, comparing the COVID patients to the non-COVID patients during the same time may not be the best comparison. It would be interesting, if possible, to compare the COVID-19 patients admitted during the study period to patients admitted the year prior in terms of rates of MV, HD, tracheostomy, LOS at originating hospital, etc. This would provide broader context for what is truly COVID related.\nMinor Comments\nIn the Abstract it is stated “Median age: 68 [44-94] years, 61% male, 80.5% with tracheostomy, 51.2% on invasive mechanical ventilation, 22% receiving hemodialysis”. It is confusing as to whether 80.5% of the 41 patients had a tracheostomy or 80.5% of patients who were receiving MV had a tracheostomy. Please clarify.\n\nIn the Methods it states “Missing data were omitted from analysis. For the variables, serum albumin and serum glucose, n=36 and n=40 respectively.” I am not sure what is meant by these sentences. Please clarify.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6568", "date": "20 Apr 2021", "name": "Meg Hassenpflug", "role": "Author Response", "response": "We kindly thank the Reviewer for his thoughtful comments and suggestions for our paper. Our responses follow: Major Comments We absolutely agree that what happens to these patients (outcomes) is of foremost interest. Referring to the study inclusion dates, this group of patients represents the beginning of the COVID pandemic in southern California. Our central purpose at this relatively early stage of the pandemic was to quickly share with critical care providers the characterization of the population of post-COVID infection patients admitted to our facility, to underscore the important role of the LTAC in responding to the COVID-19 crisis. It was not our intent to present outcomes at this early timepoint. With a small cohort of patients, and only 14 patients admitted for ventilator weaning, we believe that attempting to present any outcomes data may have been misleading. We agree that it would be helpful to understand patients’ functional status relative to a variety of specific outcomes (our interpretation of the phrase “likelihood of recovery”). These data were not included in the initial characterization of the population as it was not our intent to present any outcomes analysis at this early timepoint. The Reviewer’s point on the COVID vs non-COVID patient populations is well taken. The only minor comparison mentioned in the text between the two groups was length of stay (LOS) at the transferring hospital. An oversight on our part in the Abstract and Results is that we failed to include those data. We regret the omission and have excluded the related statement from the Abstract, and added the data to Results in the revised text. The LOS at the transferring acute care hospital was median 42 [8-78] days for the post-COVID pneumonia cohort, and median 16 [1-96] days for non-COVID patients admitted during the same time period. We made no attempt to interpret the data, and considered this simply an interesting informational contrast to be made at that point in time. We very much appreciate your suggestion to compare the COVID-19 patients admitted during the study period to patients admitted the year prior in terms of rates of MV, HD, tracheostomy, LOS at originating hospital, etc, and are doing so as we build on this effort. Thank you. Minor Comments In the Abstract, the Reviewer asks for clarification regarding whether 80.5% of the 41 patients had a tracheostomy or 80.5% of patients who were receiving MV had a tracheostomy. The percentages refer to the entire cohort, but we see where confusion could arise. The Abstract has been edited for clarity. The Reviewer asks for clarification of statements regarding missing data and the various “n” for the laboratory values serum albumin and serum glucose. We were informing the reader that these two variables presented in Table 2 were not reflective of the entire cohort of 41 patients due to a few missing values. The text has been revised." } ] }, { "id": "77537", "date": "15 Feb 2021", "name": "Anil Makam", "expertise": [ "Reviewer Expertise LTACHs", "health services research", "comparative effectiveness", "geriatrics", "post-acute care", "policy." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors conducted a straightforward small descriptive study of COVID-19 patients, mostly who had CCI, who were transferred to a single non-profit LTACH in Southern California. The report describes baseline sociodemographic details and clinical characteristics at the preceding transferring hospital and upon admission. This small descriptive cohort study provides novel data that a small but meaningful population of COVID-19 patients experience CCI from COVID-19, and implies (but does not show) that they likely will suffer from post-ICU syndrome even after their LTACH stay. I have a few comments:\nAs the pandemic evolved, hospitals and clinicians gained experience in caring for this population. Were patients transferred later during the study period different that patients transferred earlier on?\n\nPlease provide the median LOS for non-COVID patients to support the claim that the preceding hospital LOS for COVID-19 patients was twice that of non-COVID-19 patients. Also, the interpretation of this is unclear, as COVID-19 patients may not have been transferred due to unfamiliarity with the disease course and isolation requirements, and not necessarily because of differences in severity/complexity of illness\n\nWhat data were missing - just albumin and glucose? The sentence is awkwardly written, please correct.\n\nWhy were only 14 of 21 patients on invasive MV admitted for weaning? Were the other 7 chronically ventilated?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6569", "date": "20 Apr 2021", "name": "Meg Hassenpflug", "role": "Author Response", "response": "We kindly thank the Reviewer for his thoughtful comments and suggestions for our paper. Our responses follow: The Reviewer notes that as the pandemic evolved, hospitals and clinicians gained experience in caring for this population. Were patients transferred later during the study period different that patients transferred earlier on? Referring to the study inclusion dates, our paper reflects admissions during roughly a four month time period. In view of the brief snapshot in time, along with a small cohort of patients we did not attempt to compare time periods at this point. We agree that such a comparison would be of interest and we will explore three, four, and six month time intervals as we build on this effort. An oversight on our part in the Abstract and Results is that we failed to include the actual data on length of stay (LOS) at the transferring acute care hospital. We regret the omission and have excluded the related statement from the Abstract, and added the data to Results in the revised text. The LOS at the transferring acute care hospital was median 42 [8-78] days for the post-COVID pneumonia cohort, and median 16 [1-96] days for non-COVID patients admitted during the same time period. We made no attempt to interpret the data, and considered this simply an interesting informational contrast to be made at that point in time. Regarding missing data: The Reviewer notes that the statements regarding missing data and the various “n” for the laboratory values serum albumin and serum glucose were perhaps awkwardly written. We were informing the reader that these two variables presented in Table 2 were not reflective of the entire cohort of 41 patients due to a few missing values. The text has been revised. The Reviewer observes that just 14 of the 21 patients on invasive mechanical ventilation were admitted for weaning. He poses the question on whether the other seven patients were chronically ventilated. We welcome the question and thank the Reviewer for his observation. None of the seven patients excluded from weaning were chronically ventilated prior to admission to the transferring hospital. Upon evaluation by the consulting pulmonologist on admission to our LTAC, patients were determined not to be weaning candidates for the following reasons: physiologic instability (unmet readiness to wean parameters), poor mentation or neurocognitive disorders. We have now included this information in the revised text. Thank you so much for the question." } ] } ]	1	https://f1000research.com/articles/9-1241
https://f1000research.com/articles/10-304/v1	19 Apr 21	{ "type": "Research Article", "title": "Change in the criteria for hypertension based on a Peruvian population: a study based on the recent American Heart Association/American College of Cardiology guidelines", "authors": [ "Christian R Mejia", "Virgilio E. Failoc-Rojas", "Mariela Vargas", "Matlin M Cárdenas", "Armando Miñan-Tapia", "Rodrigo Aguilar", "Ricardo Correa", "Christian R Mejia", "Mariela Vargas", "Matlin M Cárdenas", "Armando Miñan-Tapia", "Rodrigo Aguilar", "Ricardo Correa" ], "abstract": "Introduction: The American Heart Association/American College of Cardiology has published new guidelines for hypertension in 2017. These guidelines change the criteria to classify patients with hypertension. There has not yet been a study that analyzes the consequences of these changes in developing countries. The objective of our study was to characterize changes in the classification of hypertension with the new guidelines among Peruvian patients and to determine the factors associated with being newly diagnosed as hypertensive. Methods: An analytical cross-sectional investigation was carried out, which was based on the secondary analysis of the data of hypertension. We compared certain variables including using antihypertensive medication, region, and other sociodemographic characteristics. We performed a descriptive analysis of the variables shown as frequency and percentage. P values that were less than 0.05 were considered statistically significant, obtained using a multivariate analysis. Results: Of the 5,615 patients in this cohort, with the old criteria 4,915 (87.5%) did not have the diagnosis of hypertension; however, with the new criteria 1,415 (25%) will be diagnosed with hypertension. 467 (9%) of patients who were not taking medications and 85 (32%) of patients who were taking medications will switch from having high-normal blood pressure to grade 1 hypertension, with the new guideline. The recent diagnosis of hypertension was associated with sex (p <0.001), age (p <0.001), being overweight (p <0.001), physical activity (p = 0.010), smoking (p = 0.031), and a history of diabetes mellitus (p <0.001). When adjusted for pharmacotherapy, only sex (p <0.001), age (p <0.001), being overweight (p <0.001), and physical activity (p = 0.001) remained statistically significant. Conclusions: The new criteria increased the percentage of patients that now will be classified with hypertension. This should be accompanied by effective politics in education, surveillance, and adequate treatment of the health care system.", "keywords": [ "hypertension", "diagnosis", "multicentric", "Peru", "guidelines" ], "content": "Introduction\n\nMultiple reports have shown that hypertension (HTN) remains one of the most important chronic diseases,1–3 despite the fact that there are increasingly better diagnostic and treatment methods.4 The current worldwide prevalence is 30%, according to different regional studies.5 Worldwide, a greater morbimortality have been reported due to the same diseases or pathologies derived from it.6–8 Multiple institutions have expressed their concern to generate changes that have a positive impact on the control of this disease.9\n\nIn November 2017, the American Heart Association (AHA) in conjunction with the American College of Cardiology (ACC) made one of the most important changes of the last decades in this subject.10 In these changes, the cut-off point for the diagnosis of hypertension is reduced, which generates a new global panorama for the diagnosis, treatment, and overall control of HTN.11 This has not yet been evaluated in prospective studies in developing countries, but it is important to determine how much the changes that have been established can affect the populations so that efforts to analyze data that can give an idea of the change that may occur. The aims of this study are to characterize changes in the classification of hypertension with the new guidelines among Peruvian patients and to determine the factors associated with being newly classed as hypertensive.\n\n\nMethods\n\nAn analytical cross-sectional investigation was carried out, which was based on the secondary analysis of the data; this is because the primary data was used to describe and find associations of chronic pathologies and others related to blood pressure in various populations of Peru.12,13\n\nThe population studied were patients who attended the outpatient clinic of hospitals in 10 Peruvian cities: belonging to the coast; Piura (29 meters above sea level (masl)), Chiclayo (29 masl), Lima (101 masl); to the mountains: Huánuco (1894 masl), Huancayo (3249 masl), Cajamarca (2720 masl), Cusco (3399 masl), Puno (3827 masl), and Cerro de Pasco (4338 masl) and to the jungle; Loreto (104 masl). These cities are of the three natural regions (coast, mountains, and jungle) and are the most important/large in the whole territory (north, center, and south). A non-random sampling was taken, for which all the patients who had systolic and diastolic blood pressure measurements in the primary base were included. Subjects were not excluded because they all had the information required for the analysis.\n\nWe included patients of major age who agreed to participate in the study and signed the informed medical consent, excluding those who had acute symptoms that alter the hemodynamic balance (fever, diarrhea, vomiting), pregnancy, any anatomical defects or surgical procedures that could potentially interfere with the proper access to blood flow to the upper arm, patients that lived less than half a year in the city where the clinic was located, and patients who had not adequately answered major questions or answered in repetitive patterns.\n\nThe main variable was the diagnosis of hypertension, this was obtained according to the two categorizations (the old and the new guidelines criteria).5,10 This was based on the measurements of systolic and diastolic blood pressure, reporting elevated once or twice, which was performed in the primary population. These primary measurements were made in triage and outpatient settings of public hospitals located in the host cities. They were performed by trained medical students, who used calibrated equipment and who were guided by international recommendations for blood pressure measurements.14\n\nThe secondary variables were those that used antihypertensive medication (according to the antihypertensive medication intake on the day the patient went to perform their medical consultation), gender (male or female), age (taken quantitatively according to the years completed), being overweight or obese (according to the body mass index, taking weight and height was also done at the time of the survey and with triage teams), physical activity (taken as yes or no, according to the self-report of the patients), smoking (taken as yes or no, according to the self-report of the patients), diabetes mellitus (according to the diagnosis of a medical professional), and the natural region where they lived (Coast: Lima, Piura, Chiclayo; Mountain: Cajamarca, Huanuco, Huancayo, Cerro de Pasco, Cusco, Puno and the jungle: Loreto).\n\nThe first step was to make the selection of the data that would be used for the study, for which a process of selection and purification of the information that had the primary base was carried out. Then we proceeded to build a database in Microsoft Excel. After that, the data was transferred to the STATA program v.12.1 (Stata Corp, Texas, USA).\n\nDescriptive statistics were generated, for which a chart of the diagnosis of hypertension was made according to having two, one or no high blood pressure and taking into account the diagnostic criteria (the previous or the current one generated by the AHA/ACC guidelines). After that, two tables were generated (one for systolic pressures and the other for diastolic ones) where the percentages of the old and new HTA categories can be seen; These tables were divided according to whether the patients had taken antihypertensive medication the same morning they were interviewed.\n\nFinally, a bivariate and multivariate analysis table was prepared, in which the outcome variable would be the new hypertensive being, defined as those that were not hypertensive with the prior criteria but that now became so; this variable was contrasted versus gender, age, being overweight, the report of regular physical activity, smoking, history of diabetes mellitus, region and adjusted by the one who uses antihypertensive medication. To obtain the crude prevalence ratios (cPR), adjusted prevalence ratios (aPR), 95% confidence interval (CI), and p-value, the generalized linear models were used, with the Poisson family, the log link function, and robust models. P-values that were less than 0.05 were considered statistically significant.\n\nResearch ethics principles were followed. Patient identification data was never used, and the primary project was approved by an IRB; endorsed by the Peruvian Institute of Health - National Hospital San Bartolome (N °: 2845-DG-OADI-N ° 822-HONADOMANI-SB-2014). The ethical principles of data use were respected.\n\n\nResults\n\nAll patients in the study were eligible and provided informed consent. Using the 2017 AHA/ACC BP guidelines criteria, from the 5,615 patients in this study, 3,724 (66.3%) will not have a diagnosis of hypertension, 476 (8.5%) will continue to have hypertension and 1,415 (25.2%) will be newly diagnosed with hypertension. Figure 1 showed that before the new guidelines 6% and 2% of the patients had HT due to one (SBP or DBP) or two (SBP and DBP) elevated blood pressures, respectively. With the new criteria, the numbers of hypertensive patients will increase to 24% and 10% for having one or two elevated BP, respectively. This means a 300% increase in hypertensive patients in this cohort.\n\nHT: Hypertension.\n\nIn this study, following the prior guidelines, 4,915 (87.5%) patients were non-hypertensive. Of them, 94.5% were classified as normal BP. With the new classification, this percentage will decrease to 71.13%. More details are shown in Figure 2.\n\nHT: Hypertension.\n\nThe 8.7% (467) of the patients that didn’t receive pharmacological treatment and 31.8% (85) of the patients that were using medication change from having a normal-high systolic blood pressure to have HT stage 1. The majority of the patients with no treatment (87.9%) switch from having blood pressure normal to normal-high. This change was less dramatic in the patients that received antihypertensive medication, only 45.7% stay in the non-hypertensive range (Table 1).\n\nThe 22.8% (1,218) of the patients that were not receiving medication and 34.1% (81) of the patients that were receiving medication switched from having normal high diastolic pressure to have stage 1 HT with the new guidelines (Table 2).\n\nBivariate model showed that being newly diagnosed with hypertension was associated with gender (p<0.001), age (p<0.001), being overweight (p<0.001), physical activity (p=0.010), smoking (p=0.031), and history of diabetes mellitus (p<0.001) when it was adjusted by region and pharmacological treatment. In the multivariate analysis, we found that age (p<0.001) and being overweight (p<0.001) increased the risk of being diagnosed with hypertension using the new guidelines criteria. In contrast, females (p<0.001) and intense physical activity (p<0.001) were associated with less risk. All of these variables were adjusted for smoking, history of diabetes mellitus, region, and pharmacological treatment (Table 3).\n\n\nDiscussion\n\nIn general, in our cohort following the 2017 ACC/AHA criteria, the patients were four times more likely to be newly diagnosed with hypertension than following the prior guidelines. This new classification has a cut off of 130/80 compared with the prior cut off of 140/90.15 This will bring a lot of burden to the Peruvian health system and other countries from the region because this increase in hypertensive patients has to be in accordance with new policies, acquisition of a bigger number of diagnostic equipment, increase of antihypertensive medications, increase on lifestyle modification, and increase of education to the newly diagnosed.16 Some of this change has been in place in some regions17 with the goal of preventing this disease but now they need to be evaluated faster by the health authorities for an immediate implementation.\n\nOne to two out of 10 patients that were not taking any antihypertensive medication switched from being non-hypertensive to being newly diagnosed hypertensive, following the systolic and diastolic new criteria. This new percentage should be captured by the health care system and education to this population should be given immediately. This creates a big problem because, in the past, this population believed that they didn’t have elevated blood pressure.15 The recruitment process of the newly diagnosed hypertensive patients should be very strict and accurate. New diagnostic techniques and treatment should be implemented.15\n\nIn the group of patients that were already using antihypertensive medication, three out of 10 patients went from being treated and having the blood pressure normal to be not adequately treated (blood pressure not at goal). All of this new data is very important because pharmacological treatment has new goals in each group of patients.18 With these new guidelines, groups that in the past were under control now can be poorly treated or not in control.19\n\nThe changes in the percentage of frequency for the new guidelines have had a high impact, of which it is recommended to study the possible new impacts of this change, with the groups that now enter the criteria of hypertension and how they influence other cardiological risks.\n\nStudies in the US have determined a change in the prevalence of arterial hypertension, with an increase in prevalence from 13.7%20 to 30.4%21 considering the JNC7 guidelines and the new ACC/AHA guidelines.\n\nThis is a topic that each Peruvian region should address because it will generate a new surveillance system, prevention intervention, and treatments in patients that were already diagnosed with hypertension. We are predicting that there will be a short-term increase in the expenditure of the healthcare system22,23 but this event will improve the public health system in the long term. There is a lot of good evidence that showed that investing in prevention strategies produce direct and indirect saving.4,22\n\nWe also found that older males classed as overweight and with poor physical activity have higher risks in becoming hypertensive with these new guidelines. These risk factors have been demonstrated before in other studies.24,25 The decision-making personnel should conduct surveillance of these risk factors and other specific ones for each specific population.\n\nOne of the limitations of this study includes information bias because this study was not designed for these specific goals. Another limitation is that the results cannot be extrapolated to all the Peruvian population due to the type of sampling that was done in the study. Despite all these limitations, this research is very important because it was done on a large cohort of Peruvian patients that participated to understand the physiological measures of blood pressure and other chronic comorbidities. This study showed the burden that this new change will cause on the healthcare of similar populations.\n\n\nConclusions\n\nOur conclusions are that with the 2017 AHA/ACC criteria, there will be more than a 300% increase in newly diagnosed hypertensive patients. This increase is less dramatic in patients that didn’t have the disease using the prior guidelines but required special attention in patients that already had the disease and were treated with antihypertensive medication. Gender, age, being overweight and poor physical activity were associated with the new diagnosis of hypertension.\n\n\nConsent statement\n\nWritten informed consent for publication of the patients’ details and/or their images was obtained from the patients.\n\n\nData availability\n\nZenodo: Change criteria hypertension Peru, data. https://doi.org/10.5281/zenodo.4567767.26\n\nThis data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nAdeloye D, Basquill C: Estimating the Prevalence and Awareness Rates of Hypertension in Africa: A Systematic Analysis. PLoS One. 2014; 9(8): e104300. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMills KT, Bundy JD, Kelly TN, et al.: Global Disparities of Hypertension Prevalence and Control: A Systematic Analysis of Population-based Studies from 90 Countries. Circulation. 2016; 134(6): 44–50. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSarki AM, Nduka CU, Stranges S, et al.: Prevalence of Hypertension in Low- and Middle-Income Countries. Medicine (Baltimore). 2015; 94(50): e1959. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGu D, He J, Coxson PG, et al.: The Cost-Effectiveness of Low-Cost Essential Antihypertensive Medicines for Hypertension Control in China: A Modelling Study. PLoS Med. 2015; 12(8): e1001860. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJames PA, Oparil S, Carter BL, et al.: Evidence-Based Guideline for the Management of High Blood Pressure in Adults: Report From the Panel Members Appointed to the Eighth Joint National Committee (JNC 8). JAMA. 2014; 311(5): 507–20. PubMed Abstract \| Publisher Full Text\n\nMoon JY, Park KJ, Hwangbo Y, et al.: A Trend Analysis of the Prevalence, Awareness, Treatment, and Control of Hypertension by Age Group. J Prev Med Pub Health. 2013; 46(6): 353–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlosco ML, Gunstad J, Xu X, et al.: The Impact of Hypertension on Cerebral Perfusion and Cortical Thickness in Older Adults. J Am Soc Hypertens JASH. 2014; 8(8): 561–70. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBosu WK: The prevalence, awareness, and control of hypertension among workers in West Africa: a systematic review. Glob Health Action. 2015; 8: 26227. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDefo BK, Mbanya JC, Tardif J-C, et al.: Diagnosis, Prevalence, Awareness, Treatment, Prevention, and Control of Hypertension in Cameroon: Protocol for a Systematic Review and Meta-Analysis of Clinic-Based and Community-Based Studies. JMIR Res Protoc. 2017; 6(5): e102. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCifu AS, Davis AM: Prevention, Detection, Evaluation, and Management of High Blood Pressure in Adults. JAMA. 2017. [cited 2017, Nov 21].Publisher Full Text Reference Source\n\nKirk JK, Allsbrook J, Hansell M, et al.: A systematic review of hypertension outcomes and treatment strategies in older adults. Arch Gerontol Geriatr. 2017; 73(S.C): 160–8. PubMed Abstract \| Publisher Full Text\n\nMejia CR, Verastegui-Díaz A, Quiñones-Laveriano DM, et al.: Actividad física y su asociación con enfermedades crónicas en ancianos de 11 ciudades del Perú. Gac Med Mex. 2017; 153: 482–487. Publisher Full Text\n\nMejia CR, Failoc-Rojas VE, So E, et al.: Characteristics and Factors Associated With Antihypertensive Medication Use in Patients Attending Peruvian Health Facilities. Cureus. 9(2): e1011. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOrganización Mundial de la Salud: Guidelines for training and practical instruction. Section 4: Vigilancia STEPS de la OMS. 3-4-14. [Internet]. [cited 2017, Dec 3]. Reference Source\n\nWhelton PK, Carey RM, Aronow WS, et al.: Guideline for the Prevention, Detection, Evaluation, and Management of High Blood Pressure in Adults: A Report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines. J Am Coll Cardiol. 2017 [In press]. [cited 2017, Dec 3]. Reference Source\n\nMancia G, Rea F, Cuspidi C, et al.: Blood pressure control in hypertension. Pros and cons of available treatment strategies. J Hypertens. 2017; 35(2): 225–233. PubMed Abstract \| Publisher Full Text\n\nPatel P, Ordunez P, DiPette D, et al.: Improved Blood Pressure Control to Reduce Cardiovascular Disease Morbidity and Mortality: The Standardized Hypertension Treatment and Prevention Project. J Clin Hypertens. 2016; 18(12): 1284–94. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCampos K, Sheth S, Coulter SA: Hypertension Treatment ACCORDing to SPRINT. Tex Heart Inst J. 2016; 43(4): 324–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMuntner P, Carey RM, Gidding S, et al.: Potential US Population Impact of the 2017 American College of Cardiology/American Heart Association High Blood Pressure Guideline. Circulation, CIRCULATIONAHA-117. [cited 2017, Dec 3]. Reference Source\n\nMuntner P, Carey RM, Gidding S, et al.: Population Impact of the 2017 ACC/AHA High Blood Pressure Guideline. J Am Coll Cardiol. 2018 Jan 16; 71(2): 109–118. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVaduganathan M, Pareek M, Qamar A, et al.: Baseline Blood Pressure, the 2017 ACC/AHA High Blood Pressure Guidelines, and Long-Term Cardiovascular Risk in SPRINT. Am J Med. 2018 Feb 5; pii: S0002-9343(18)30097-4. PubMed Abstract \| Publisher Full Text\n\nBress AP, Bellows BK, King JB, et al.: Cost-Effectiveness of Intensive versus Standard Blood-Pressure Control. N Engl J Med. 2017; 377(8): 745–55. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWebb M, Fahimi S, Singh GM, et al.: Cost effectiveness of a government supported policy strategy to decrease sodium intake: global analysis across 183 nations. BMJ. 2017; i6699: 356. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZubeldia LL, Quiles IJ, Mañes VJ, et al.: Prevalencia de hipertensión arterial y de sus factores asociados en población de 16 a 90 años de edad en la Comunitat Valenciana. Rev Esp Salud Publica. 2016; 90(1): e40006.\n\nIsmael C-N, Lucía H-B, Rosalba R-M, et al.: Hipertensión arterial: prevalencia, diagnóstico oportuno, control y tendencias en adultos mexicanos. Salud pública Méx. 2013; 55(S2): 144–150.\n\nVirgilio F-R: Change criteria hypertension Peru, data [Data set]. Zenodo. 2021. Publisher Full Text" }	[ { "id": "93898", "date": "27 Sep 2021", "name": "José Fernando Gómez Montes", "expertise": [ "Reviewer Expertise geriatric medicine. internal medicine. falls. epidemiology." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a cross-sectional study based on the secondary analysis of the data from patient surveys in health facilities in the 10 cities of Peru. The aim “To examine the impact of the new 2017 ACC/AHA hypertension guideline on the prevalence of hypertension and their factors associated. The topic of the manuscript is appropriate for the Journal. It could be of interest to investigators and clinicians. However, several essential revisions are necessary\nMajor compulsory revisions\n\nFirst question… why authors no included in the introduction or discussion section an identical paper published in Spanish in Rev Med Chile in 2019:\nHernández-Vásquez A, Rojas-Roque C, Santero M, Ruiz-Maza JC, Casas-Bendezú M, Miranda JJ. ¿Qué representa cambiar el umbral diagnóstico de la hipertensión arterial? Guías ACC/AHA 2017 y su aplicación en Perú [Changes in the prevalence of hypertension in Peru using the new guideline of the American College of Cardiology]. Rev Med Chil. 2019 1\n\nRegarding Guidelines ACC/AHA 2017 authors mentioned “This has not yet been evaluated in prospective studies in developing countries”, however, several papers in low and middle income countries (LMICs) have been published. Please check at least these papers:\n\nKibria GMA, Swasey K, Kc A, Mirbolouk M, Sakib MN, Sharmeen A, Chadni MJ, Stafford KA. Estimated Change in Prevalence of Hypertension in Nepal Following Application of the 2017 ACC/AHA Guideline. JAMA Netw Open. 2018 Jul 6;1(3):e180606. doi: 10.1001/jamanetworkopen.2018.0606. PMID: 30646022; PMCID: PMC6324293.2\n\nRahman MA, Halder HR, Yadav UN, Mistry SK. Prevalence of and factors associated with hypertension according to JNC 7 and ACC/AHA 2017 guidelines in Bangladesh. Sci Rep. 2021 Jul 29;11(1):15420. doi: 10.1038/s41598-021-94947-2. PMID: 34326418; PMCID: PMC8322062.3\n\nKibria GMA, Swasey K, Choudhury A, Burrowes V, Stafford KA, Uddin SMI, Mirbolouk M, Sharmeen A, Kc A, Mitra DK. The new 2017 ACC/AHA guideline for classification of hypertension: changes in prevalence of hypertension among adults in Bangladesh. J Hum Hypertens. 2018 Sep;32(8-9):608-616. doi: 10.1038/s41371-018-0080-z. Epub 2018 Jun 13. PMID: 29899377; PMCID: PMC6487869.4\n\nLi D, Zeng X, Huang Y, Lei H, Li G, Zhang N, Huang W. Increased Risk of Hypertension in Young Adults in Southwest China: Impact of the 2017 ACC/AHA High Blood Pressure Guideline. Curr Hypertens Rep. 2019 Feb 28;21(3):21. doi: 10.1007/s11906-019-0926-y. PMID: 30820764.5\nWhy authors non included a table to show characteristics of participants?. This is strongly recommended. The discussion section should include comments and comparisons with the papers cited above. Moreover, a comment comparative with Mejia and cols. (2017) paper is desirable. More information is needed in the discussion section about findings in other LMICs.\n\nMinor essential revisions:\nTittle:\nThe title is accurate and sufficiently descriptive of the content.\n\nAbstract: Abstract: Must be changed, due there are at least 5 papers with the same aim in LMICs Secondary analysis of the data of hypertension is no clear for the reader. Please revise.\nIntroduction: Another concern about this paper is why it is a prospective study mentioned in the introduction section, however, in study design “an analytical cross-sectional” is referred to? Please clarify this confusion.\n\nMaterial and methods: Please clarify why the study referenced as 12 is based in primary analysis?. More information about what was exactly his aim, more than only “find associations of chronic pathologies related with blood pressure”. Why the data based referenced as primary analysis included only data of the 894 patients with hypertension, while this research for secondary analysis mentioned 5.615 participants? Please clarify this situation. “Subjects were not excluded because they all had the information required” should be interpreted as missing data were included?. Please clarify this important bias in this paper.\nVariables description should be more clear, for example, Physical activity, what tools were used? How blood pressure was taken?. Please check Kibria y cols. (2018). Definition of hypertension should be mentioned in the material section for more understanding of readers. Only cut-off levels are commented in the discussion section\nResults: Information is clearly provided.\nDiscussion Please check above.\nReferences: There were 26 and all are appropriate, however, non important references about the topic in LMICs were included. Check above.\nTables and figures: Three tables and two figures are shown. All are well presented. However, an initial table with sample description is recommended.\nThanks for letting me review this manuscript. This could be a nice paper.\nLevel of interest: An article whose findings are important to those with closely related research interests. Quality of written English: Well. Statistical review: No. Declaration of competing interests: I declare that I have no competing interests.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "144896", "date": "09 Aug 2022", "name": "Saman Khalesi", "expertise": [ "Reviewer Expertise Hypertension", "epidemiology", "health behaviour" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAbstract\nThe methods section needs clarification on how the authors identified “the factors associated with being newly diagnosed…” - what “multivariate analysis” was used as there are many types.\n\nResults section: If the significant p-value is defined in the methods, what is the use of reporting p values in the results? Instead, I suggest reporting coefficients (aPR etc.) or risk/likelihood which provides a better understanding of the modelling.\n\nConclusion section: I believe it should read as effective policies in education instead of “politics in education”.\nIntroduction\nI am not sure I follow the significance of the study, “…but it is important to determine how much the changes that have been established can affect the populations so that efforts to analyze data that can give an idea of the change that may occur” - what do the authors mean by the last part? Isn’t the aim of the study to focus on the Peruvian population to call for interventions and policies to help prevent and manage HTN in this population? If so, please consider changing the abstract to also reflect this.\n\nWhat is the significance of identifying factors associated with being newly diagnosed? I think this also goes back to interventions and policies targeting the at-risk population. The significance of the study needs rewording.\nMethods\nI don’t think the second half of the “study design” section adds anything to this paper, what is the use of knowing what the primary data was used for?\n\n“Non-random sampling” does this refer to selective sampling? What was the reason of excluding patients who lived less than half a year in the city?\n\nI don’t think the first sentence of data analysis is required, instead better explanation of the GLM could help.\nResults\nPlease include the characteristics of the participants. Some sections need rewording, for example, “This means a 300% increase in hypertensive patients in this cohort” - maybe change to “This means a 300% increase in hypertensive diagnosis using the 2017… guidelines”.\nConclusion.\n“This increase is less dramatic in patients that didn’t have the disease using the prior guidelines…” - I'm not sure what results this conclusion refers to. If anything, it should suggest the findings to be more dramatic for this cohort\nI highly recommend proof-reading the manuscript before the next submission as some sentences and paragraphs need rewording/restructuring.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-304
https://f1000research.com/articles/10-302/v1	19 Apr 21	{ "type": "Opinion Article", "title": "Developing open-source software for bioimage analysis: opportunities and challenges", "authors": [ "Florian Levet", "Anne E. Carpenter", "Kevin W. Eliceiri", "Anna Kreshuk", "Peter Bankhead", "Robert Haase", "Anne E. Carpenter", "Kevin W. Eliceiri", "Anna Kreshuk", "Peter Bankhead", "Robert Haase" ], "abstract": "Fast-paced innovations in imaging have resulted in single systems producing exponential amounts of data to be analyzed. Computational methods developed in computer science labs have proven to be crucial for analyzing these data in an unbiased and efficient manner, reaching a prominent role in most microscopy studies. Still, their use usually requires expertise in bioimage analysis, and their accessibility for life scientists has therefore become a bottleneck. Open-source software for bioimage analysis has developed to disseminate these computational methods to a wider audience, and to life scientists in particular. In recent years, the influence of many open-source tools has grown tremendously, helping tens of thousands of life scientists in the process. As creators of successful open-source bioimage analysis software, we here discuss the motivations that can initiate development of a new tool, the common challenges faced, and the characteristics required for achieving success.", "keywords": [ "Open-source", "software", "bioimage analysis", "life science" ], "content": "Introduction\n\nModern imaging techniques1–5 have brought microscopy into a computationally-intensive era by capturing biological systems at a level of spatial and temporal resolution never achieved before.6 A modern imaging system can easily generate terabytes of data for a single acquisition, and it is not uncommon for a biological study to involve dozens of samples from several different conditions. This massive amount of data has driven the adoption of new computational methods at all steps involved in imaging, whether it be for image acquisition,7,8 restoration,9 data storage10 or real-time processing.11 Bioimage analysis has therefore become a crucial step integrating data science techniques into life science. Yet, the multiplicity of computational solutions has shifted the bottleneck: many of these solutions require substantial computational expertise and customization, limiting their use among many life scientists.\n\nThis expertise issue has been partly addressed by the emergence of specialists known as bioimage analysts. Originating from diverse backgrounds, such as computer science, microscopy and life sciences, they exhibit an overall understanding of many steps involved in life science experiments, spanning from biology to microscopy and analysis.12 Acting as bridge-builders between life scientists and computer scientists, bioimage analysts develop skills to identify and adapt the best available computational solutions to a specific biological problem. Often employed in core facilities, their task is to deliver tools and workflows to end users - typically in biology or biophysics oriented labs - which go beyond service provision into experimentation and innovative technology development.13 Depending on the analysis task, a workflow can require several tools, often split among multiple software libraries and even programming languages, to be identified, combined, customized and optimized to properly address a specific scientific question. However, many biologists still do not have access to core facilities employing such bioimage analysts and lack the in-depth knowledge, experience, and time required to develop, validate and apply such sophisticated workflows.\n\nThus, an important challenge is to directly provide life scientists with comprehensive, fully-featured and easy-to-use tools for domain-specific quantitative measurements that they can execute themselves. This development effort has been taken on by a subset of bioimage analysts, usually computer scientists by training, who develop open-source software platforms to improve the accessibility of computational methods for both “power users” and biologists with very little computational training. The resulting open source image analysis platforms have achieved great success and had a long-lasting impact on life science: the ability to access analysis methods’ source code have accelerated their adoption, improved reproducibility and facilitated the combination and implementation of new methods. Nevertheless, anyone seeking to develop biology-oriented open-source software platforms faces numerous challenges in terms of packaging, documentation, maintenance, reproducibility and funding. In this opinion paper, we intend to discuss these aspects in a way that will be beneficial both for developers and users. Developers will be exposed to minimally required motivations that justify the creation of a new open-source tool, as well as best practices to follow during development. Users will be introduced to some of the challenges faced by developers, hopefully leading to a better understanding of the open-source bioimage analysis ecosystem, and how they might personally contribute to the development and support of the software tools they need.\n\n\nChoosing the software’s scope and audience\n\nThe development of a new bioimage analysis software platform is often motivated by the desire to answer a specific unmet biological question. However, bringing a broadly useful tool to users is a substantial commitment that typically requires many person-years of work (Table 1). It is important that this is undertaken with an understanding of the specific challenges and opportunities, to reduce the risk of a promising project later being abandoned and leaving users unsupported. A crucial first question is to define the scope and intended audience for the software.\n\nAt one end of the spectrum, one can simply write the minimum code necessary to address a biological question, and provide scripts alongside the paper publishing the results. This is a perfectly acceptable route and should be considered for cases where the image analysis solution is heavily customized to the problem and thus of limited use to others, or where there is no strong prospects of future support, maintenance, and development. We should applaud efforts to still make code available, for reproducibility reasons, even when it is neither polished nor broadly useful.14\n\nAt the other end of the spectrum, one can launch an entirely new software effort around the code needed for a particular project, expanding this to serve a broader audience over time. Here, a crucial consideration is whether there is a plausible strategy for future support, maintenance, and development in the long term, especially if the software is widely adopted and therefore becomes relied upon by a community of scientists. A successful project may grow far beyond the initial expectations of the creator, and as this happens the nature of the required work tends to change (Figure 1). In the words of Kurt Vonnegut, “[a] nother flaw in the human character is that everybody wants to build and nobody wants to do maintenance” - or, perhaps more relevant in this context, nobody wants to fund maintenance.16\n\nCreated to solve a biological analysis need, the tool is released and published. As it is adopted by new users, the developer begins to spend time on user support and maintenance, while still managing to add new methods. Finally, as the number of users continues to grow, the developer is overwhelmed with user support and maintenance. As this stage, securing funding for new dedicated developers is crucial.\n\nAn intermediate position is to develop a plugin or extension for an existing platform. Extensibility has been key to the success of software projects such as ImageJ,17 Fiji,18 Icy19 and CellProfiler,20 with the result that many novel algorithms are developed as plugins for these platforms. One example is the CLIJ library, which brings manifold implementations of accelerated image processing algorithms as plugins to platforms such as ImageJ and Icy.11 This strategy not only reduces effort for the developer, who can focus primarily on developing new functionality beyond that already available, but also reduces the effort required by users to become familiar with entirely new softwares. For this reason, we advocate developing plugins or adapting existing solutions wherever possible. This has the added benefit of improving the robustness of the core software: reusing components results in more thorough testing and therefore fewer undiscovered bugs.\n\nNevertheless, new open source bioimaging software applications are sometimes needed as the computational and biological landscape changes over time. If a major gap is identified, as was the case for supporting and visualizing multidimensional image data,21,22 then developing a new software platform can be the best way to serve the community and lead to great success. Indeed, if the software is user-friendly and well packaged, its adoption by the community can be swift.23–28\n\nWhen launching a new software project, our experience is to carefully consider the boundaries of the audience to be served. As maintenance is an ongoing tax against future software developments (not to mention research progress, which is often necessary for funding the software), it is important to not be overly ambitious by attempting to create software that addresses too many distinct problems simultaneously. For example, in the CellProfiler project, we aimed to serve the community needing modular, automated pipelines, and left interactive manual analysis and image visualization tools to ImageJ, which was well-developed in these features. We also left a gap for very large-format images such as for pathology, which would have been difficult to support well given its underlying infrastructure; QuPath was later developed specifically to meet this need from the ground up.23 Likewise, Single-Molecule Localization microscopy was not well served by generic bioimage analysis platforms, because it produces point clouds rather than conventional images; the Tesseler-suite25,29 and its successor PoCA30 were created to meet this need. In another example, ilastik31 was developed with the aim of providing interactive machine learning tools, intentionally leaving pre- and post-processing steps to Fiji and CellProfiler. OMERO has always remained largely focused on image storage and organization.32 Defining the scope in this way prevents a project from being spread too thin and serving no audience very well. Furthermore, interoperability between software using open standards makes it possible to gain the benefits of combining applications.\n\nAnother choice to consider is whether to serve point-and-click users versus those who are comfortable writing code themselves; some software serves both ends of this spectrum well, but again defining the audience proactively ensures that developer time is not spread too thin meeting those needs. The skills and effort required to design an intuitive and responsive graphical user interface are quite distinct from those needed to devise new image analysis algorithms, and are therefore understandably not always a core focus. Furthermore, while a developer who chooses to concentrate on user-friendliness may find that their software is more widely adopted, this can be a double-edged sword in that it may dramatically increase demand for support, make delivering new functionality more onerous, and reduce the time available to focus on new projects or publications (Figure 2). Some developers consider these costs justified because they can increase the impact of their work, but ultimately it is a decision that needs to be made: even highly-technical software is a creative endeavor through which developers can express their own interests and values.\n\nThese charts only take into account paid staff developers.\n\n\nProgramming language\n\nThe choice of programming language is a core decision that has long-term impact. It is usually a trade-off between the developer’s preference and experience versus external factors, such as an entity enforcing the use of a particular language to ensure compatibility between their tools, the need to use specific libraries, or the desire to interoperate with existing tools.\n\nTraditionally, software platforms were usually developed in C/C++ or Java, but the last decade has seen the rapid emergence of Python in the scientific landscape. C/C++ can offer excellent performance and is still the dominant language in Computer Graphics, an important point when one wants to transfer this research to bioimage analysis tools, but portability can be an issue because tools developed in C/C++ need to be compiled for every targeted operating system. In contrast, Java applications are portable and can be executed on all major platforms, without the need for recompilation. Java applications can also readily use the image processing ecosystem around ImageJ17 and Bio-Formats for image file reading/writing;33 together these advantages help explain why so many bioimage analysis platforms have been developed in Java. Finally, recent years have seen an important increase in the number of workflows and tools developed in Python. Python has become the language of choice for data science and can be self-learned by non-computer scientists. Combined with its numerous available scientific libraries, Python represents a solution that is well-suited for developing new analysis methods using both image processing and machine learning techniques.\n\nEach language has its pros and cons, and bridges exist to allow communication between tools developed in different languages. Nevertheless, working across programming languages makes development and distribution of software more difficult, introducing a challenge that can be hard to bear for small-sized research groups.\n\n\nCommunities\n\nMost bioimage analysis software platforms start with a single developer and a single user, who are often the same person. Over time many more users and developers can be attracted into a rich and healthy community. There are many ways to support open software that do not require writing code. Often, the most valuable contributions can be from experienced users providing examples, writing documentation, answering questions for their peers and creating online resources, and testing new release candidates.\n\nIn open source projects, the developer community can include individuals from all over the world who modify or develop new methods and ask the authorized maintainers to merge their work within the tool’s official code. This can be extremely beneficial, enhancing the software and broadening its scope. In widely used projects the maintainers can, however, become overwhelmed by requests, which risk pulling the project in different directions without dedicated engineers determining the future vision. There is also a risk that the creator of a successful application can feel reduced to an administrative role as their project grows, with their time being taken up by reviewing the contributions of others and answering user questions instead of solving problems and writing code themselves, with diminishing recognition for this essential work. Software designed for point-and-click users will by nature have fewer coding contributors than libraries designed for computationalists’ use. Ultimately, this shows the necessity for bioimage analysis tools to have both funded developers dedicated to handling the core software and plugin mechanisms allowing individuals to develop new methods without modifying the core code.\n\nThe bioimage analysis community is extremely welcoming and friendly. The Scientific Community Image Forum (https://forum.image.sc/),34 has rapidly emerged as an essential entry point in the field, and is the preferred place for any bioimage analysis related question. It is already the primary designated discussion point for more than 40 open source tools/libraries. One of its characteristics is that it is extremely easy for a new open-source bioimage analysis tool or library to be included as a community partner. Because of its wide audience, it can tremendously benefit developers by (i) facilitating the discovery and recognition of their tools and, (ii) alleviating the burden of answering new users' questions, both because other users of the software can pitch in and because answers remain publicly available and searchable. Finally, the forum creates a network of developers in which often-isolated developers can get help for technical issues.\n\n\nPackaging\n\nMaximizing the impact of a new tool towards life scientists usually requires a well-designed Graphical User Interface (GUI) that allows users to interact with the software platform. As mentioned above, this can require considerable effort from the developer. Scientific image data are extremely variable, with the result that analysis often involves customization. It can be extremely challenging to provide a balance between user-friendliness, flexibility and extensibility within a GUI. A technical compromise can be to provide core functionality through a point-and-click interface supplemented by scripting for power users.\n\nAn easy and comprehensible installation process is just as important for adoption. For installation, traditionally, platforms developed in compiled languages (C/C++ or Java) are shipped with 1-click installers, the easiest solution possible, but requiring significant expertise from the developer. For the user, the best scenario is when all dependencies are directly included in the installer or are automatically downloaded. If they are not included, the user will have to install them separately, a potential source of issues and a support burden for the developer. Interpreted languages such as Python have slightly shifted this common process as scripts/apps can be bundled as packages and easily installed using package-management systems such as pip. In practice, dependencies or version issues are not uncommon, and installing Python modules may still require some level of expertise. Python scripts can also be bundled in web-based interactive computational environments (such as Jupyter Notebooks). To ensure simplicity, we advocate providing 1-click installers for bioimage analysis tools where feasible, supporting all three popular operating systems.\n\nThe time-consuming nature of packaging highlights another advantage of developing plugins for existing platforms: they already provide standardized GUI elements and installers that are familiar to users. Furthermore, if developers from different tools share efforts in improving and maintaining a pre-existing GUI, long-term adoption by a larger community can be achieved.\n\n\nVersion control and quality assurance\n\nThe development process of scientific software includes two more important elements: Firstly, version control is good scientific practice, allowing developers to maintain their code in a modern fashion while tracing changes and contributions. The git platform (https://git-scm.com/) is most commonly used for version control. Furthermore, releases should be tagged and archived to allow reproducing scientific results produced with a given release of a scientific software later on. Secondly, automatic and manual quality assurance procedures should be established in scientific software projects to minimize potential issues introduced when code changes. Software is nowadays highly complex and developers cannot foresee if a change may cause issues in code that depends on the modified function. Continuous integration systems can help by executing pre-defined automated tests that alert developers in case nightly builds fail because tests do not run or produce a change in outputs. Both version control and continuous integration are indisputable requirements for any software that aims to attract external contributions. Nevertheless, the best automated test system cannot replace manual testing, which should occur for critical core libraries or major releases to end users. Effort for such manual tests can be shared within the community. In our experience, power users of scientific software can be easily motivated to test a release candidate or beta version of a software because they enjoy exploring the newly introduced features and providing feedback to developers. For guiding testers during this process and to support developers who program tools that depend on a given software, release notes are highly recommended to inform the community about recent changes and what to take care of when upgrading to a newly released software version.\n\n\nDocumentation\n\nSoftware projects aiming to bring services and solutions to a broad audience require special emphasis on documentation: beyond a publication that mostly serves as an announcement of major software features, there are four kinds of modern software documentation:35 1) learning-oriented tutorials targeting beginners and introducing them to basic principles of a given software or software library; 2) goal oriented guides documenting typical use cases of a software, in which users can learn a new tool; 3) understanding-oriented discussions benefiting developers who might, especially in open-source projects, become contributors to extend and maintain a given software project; and 4) information-oriented reference manuals serving as a glossary of functionality, for example as a searchable database of individual functions to explore features and read their concise description.\n\nDocumentation should not be seen as the final step before launching a new software. It is rather a continuous process, particularly when new features are added. Furthermore, user feedback about the documentation can ultimately also lead to updates in the software. Software features that are intuitive to the developers might be complicated to explain in the user guide while writing the corresponding documentation, triggering a rethinking of how the feature is offered to the user and changing the GUI or the underlying algorithm. This will improve user experience and the documentation.\n\nFinally, newly released bioimage analysis tools should always be provided with example datasets, for example included in the installer or uploaded on online platforms, such as Zenodo (https://zenodo.org), chosen to illustrate some specific features. Additionally, complete protocols for processing these example data sets should be provided, for example via online platforms such as Protocols.io (https://protocols.io). This decreases the likelihood that users trying a new tool for the first time will experience a crash or incomprehensible analysis result. Instead, running the desired analysis method on an example dataset will help them understand how others analyze data of similar kind to theirs.\n\n\nInteroperability\n\nThe term interoperability describes how different software can be combined in common workflows or substitute each other. Interoperability is mostly achieved through two levels: (i) external interoperability by the use of common file formats or libraries, and (ii) internal interoperability through a plugin mechanism. For constructing workflows efficiently, both internal and external interoperability have advantages and disadvantages: if possible, new software should adopt both to maximize interoperability.\n\nIf a software stores processing results in common file formats such as text (txt), comma-separated values (csv) or common image file formats, it can be considered as interoperable with other software that processes such files. Software that introduces unique, proprietary and/or closed-source file formats for handling data introduces a barrier and is not interoperable with other software. Much effort has been devoted by the bioimage community to promote external interoperability. For instance, Bio-Formats33 is a software library for reading proprietary microscopy formats and converting to open standardized formats. These efforts are also resulting in community efforts towards new open formats.36,37\n\nOn a higher level, internal interoperability can be achieved by plugin mechanisms to ensure that within a given software application, modules from various origins can communicate with each other. Software such as ImageJ17/Fiji,18 CellProfiler,20 KNIME,38 or Icy19 are known in the image data science field as platforms that are extensible using plugin architectures. Such interoperability can be beneficial to all software tools involved and drive innovation. A case in point is KNIME, which wanted to add image processing functionalities to its features set and decided against developing their own from scratch. Instead, they directly used ImageJ's functionalities and, in the process, helped improve both tools.39,40 Noticeably, an important project enabling interoperability is SciJava (http://scijava.org) originally developed as part of the ImageJ ecosystem but then adopted by others;39 it provides an overview of available Java libraries for scientific computing including file format import support and data visualization.21\n\n\nLicensing\n\nLicensing is an aspect of open-source software platforms that is often overlooked by developers in spite of its importance. More often than not, the license is eventually chosen when the source code is released, whereas it should be defined from the start. Licensing is essential because it defines how others are able to use the source code of a given project.\n\nIn most countries, selecting no license at all results in copyright by the author, all rights reserved, completely working against the very notion of open-source. This prevents code reusability because it would be unsafe to use copyrighted code.\n\nA helpful resource in selecting a license is the Open Source Initiative (https://opensource.org/), which maintains a list of approved open source licenses. Basically, they can be divided in three categories: strong copyleft, weak copyleft, and permissive licenses. Strong copyleft licenses (GPLv2, GPLv3, AGPL, etc.) grant permission to use, modify, and redistribute code as long as the original license remains intact for both the original project AND any project using any code of the original project. Although at first glance this sounds like the most open possible choice, there is a catch: a project with a strong copyleft license will force any subsequent project using part of its code to be under the same copyleft license and prevents the developer from using code from projects without this same license. In contrast, weak copyleft licenses (LGPL, MPL 2.0, etc.) do not extend their protection across linkage boundaries. This essentially means that, while the original project and its modifications will retain the weak copyleft license, any code linking to it will be allowed to use a different license (even proprietary code). Finally, permissive licenses (BSD, Apache 2.0, MIT, etc.) usually only retain the attribution restriction: any derived project will require statements giving credits to the original one while still being able to use, modify and distribute it, even commercially. Several of us felt quite protective of code at the beginning of our projects, thinking that a permissive license would risk our ability to maintain a unique grant-fundable project, but over time we have seen the benefits of our code bases being used to accelerate science however they are used by others.\n\n\nPublication\n\nA project’s release may include executable software, code, documentation and tutorials; another milestone of an open-source software platform is its publication. For projects launched to solve a particular biological question, the best-case scenario is when the tool is ready to be released before the collaborators' biology study. In this case, a technical paper can present the tool and its new method without jeopardizing the biology publication; separate publication may even strengthen both.\n\nProblems may arise when the biology paper is ready first and the software must be mentioned or described in it. Indeed, most journals are driven towards novelty, and presenting a tool that “only” provides a broader access to already published methods is often considered lacking novelty. This also holds true for plugins and is detrimental to putting efforts in making computational methods truly usable. And while several journals41 may consider papers describing open-source software, managing to publish these tools in high-impact journals is highly challenging. Editors can struggle to predict the potential value and usability of a tool, especially for recent ones that have yet to reach a sizable audience. Nevertheless, we have detailed in this paper characteristics expected from a successful project; these criteria may help editors and reviewers in their assessments.\n\nAcademic labs mainly use short-term contracts. This implies that any successful open-source tool that managed to achieve long-term sustainability will hire several developers along the course of its life. As a consequence, one publication occurring at an early stage of a project will not accurately capture the whole picture of involved developers. Still, they all deserve recognition for their work and finding a way to reward them is essential. One solution is to write a new article with an updated list of authors when a new version of the project, with significant changes, is released. An ideal solution would be that the journal that first published the project would consider for peer-review this new paper, with criteria such as improved user-experience, new visualizations and methods added, etc.\n\nFinally, two main indicators are, for better or worse, used by institutions and funding agencies to evaluate an open-source project: the reputation/impact factor of the journal in which the project is published and its number of citations. Therefore, properly citing bioimage analysis is of utmost importance for ensuring their long-term sustainability. For that reason, users should cite all tools used, whether for visualization, analysis or manipulation. When a plugin of a generic bioimage analysis platform is used, both the platform- and plugin-related papers should be cited. It is currently estimated that software tools are properly cited less than half of the time.42\n\n\nFunding\n\nSoftware maintenance and support is rarely glamorous, but is one of the most cost-effective ways to impact science: the efforts of a small number of individuals can dramatically improve the efficiency and accuracy of analysis performed by thousands of researchers, helping to maximize the value of data across many studies. However, traditional research funding agencies foster innovation, and thus, typically development of new methods and tools, as opposed to maintenance. This model arose and may have been sensible for technologies other than software, which are often adopted by companies after the proof-of-principle - developed with the research funding - is successfully demonstrated. Open-source software platforms are in a strategically suboptimal position when it comes to applying for funding in this context, because novelty is rarely the goal - indeed, robustness and standardization are usually more desirable. Thus, software maintenance and long-term support can often not be funded using research grants.\n\nHowever, in recent years, exceptions have arisen: The German Research Foundation (DFG) offered funding specifically for “Research Software Sustainability”,43 the U.S. Chan Zuckerberg Initiative (CZI) has begun supporting open-source software for science generally and for bioimaging specifically44 and the National Institutes of Health (NIH) in the United States funded the Center for Open Bio-image Analysis (COBA) (https://openbioimageanalysis.org/). Likewise, the European Cooperation in Science and Technology (COST) enabled forming the Network of European Bio-Image Analysts (NEUBIAS), which enables interdisciplinary exchange between developers and end-users on a high level leading to many collaborations and new projects in the context of open-source software for biological image data science. While the mentioned funding programs offered opportunities to scientific software-focused communities, it remains challenging as an individual research group to apply for funding to turn a software-focused research project into a community-driven open-source project which is maintained long-term and serves important needs of the scientific community. We would therefore advocate more funding agencies to follow the footsteps of CZI, NIH and DFG and create programs tailored for funding maintenance and continuous development of open source tools, since they have become crucial to most biology related research.\n\n\nSummary\n\nIn just a few decades, computational methods have radically transformed the way biological images are analyzed. From tedious manual quantification, life scientists now have access to an overwhelming array of automatic and unbiased analysis tools. In this context, open source software has played a key role, offering cutting-edge analysis methods to a wide audience. The value of open source is not simply that the software is typically free of financial cost: by disclosing the source code under a recognized license, open source software enables transparency, reproducibility, standardization, and a foundation that can be built upon by others. All these aspects are increasingly important as most funding agencies move towards Open Research.\n\nDespite the great success achieved by many open source tools, developing new software from scratch should not be taken lightly. To prevent overlap with established tools, it is crucial to identify a major gap in the current landscape. The true costs of development and maintenance should be considered, and a careful long-term sustainability plan developed. If none of these conditions are met, we advocate for developing plugins to existing generic platforms, as this strongly alleviates both the maintenance issue, the activation barrier for users and the method’s overall accessibility.\n\nIf the strategy is still to develop a new tool, a few best practices should be implemented from the beginning. First, documentation should not be an afterthought, written at the time of the project’s release; it is a crucial ongoing process that should closely follow current developments. Second, version control should be embraced as it helps research reproducibility by allowing easy tagging of releases, brings intrinsic organization to a project and ensures that all contributions can be recognized. Third, any new project should select its license during its inception as it will have a profound impact on the way its code will be reusable. Fourth, GUI elements should be carefully designed to improve the project’s usability, when the audience suits it.\n\nIn spite of all these challenges, developing open source bioimage analysis software can be highly rewarding. The opportunity to durably impact a life science field by filling an analysis gap, and therefore to reach a whole new community, is a worthy motivation. Unfortunately, even the most successful open source tools still struggle to obtain funding for maintenance and continuous development. There is a clear mismatch between these difficulties and the influence of those tools that help thousands of life scientists shaping their data into quantitative measures. For that reason, we hope that funding open source tools will become easier in the future.\n\n\nData availability\n\nNo data is associated with this article.", "appendix": "Acknowledgements\n\nThis publication was supported by COST Action NEUBIAS (CA15124), funded by COST (European Cooperation in Science and Technology).\n\n\nReferences\n\nHuisken J, Stainier DYR: Selective plane illumination microscopy techniques in developmental biology. Development. 15-Jun-2009; 136(12). The Company of Biologists Ltd, pp. 1963–1975. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu Z, Lavis LD, Betzig E: Imaging live-cell dynamics and structure at the single-molecule level. Mol Cell. May 2015; 58(4): 644–659. PubMed Abstract \| Publisher Full Text\n\nSahl SJ, Hell SW, Jakobs S: Fluorescence nanoscopy in cell biology. Nat Rev Mol Cell Biol. Sep. 2017; 18(11): 685–701. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLevet F: PoCA: Point Cloud Analyst.2021. Reference Source\n\nBerg S, et al.: ilastik: interactive machine learning for (bio) image analysis. Nat. Methods. Dec. 2019; 16(12): 1226–1232. PubMed Abstract \| Publisher Full Text\n\nAllan C, et al.: OMERO: Flexible, model-driven data management for experimental biology. Nat Methods. 28-Mar-2012; 9(3). Nature Publishing Group, pp. 245–253. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLinkert M, et al.: Metadata matters: Access to image data in the real world. J Cell Biol. 31-May-2010; 189(5). The Rockefeller University Press, pp. 777–782. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRueden CT, et al.: Scientific Community Image Forum: A discussion forum for scientific image software. PLOS Biol. Jun. 2019; 17(6), p. e3000340. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nProcida D: The four kinds of documentation, and why you need to understand what they are.2017. Reference Source\n\nGonzalez-Beltran AN, et al.: Community standards for open cell migration data. GigaScience. 20-May-2020; 9(5). Oxford University Press, pp. 1–11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSwedlow JR, Gault D, Besson S, et al.: Next Generation File Formats for BioImaging.2020. Reference Source\n\nBerthold MR, et al.: KNIME: The konstanz information miner. in: 4th International Industrial Simulation Conference 2006, ISC 2006. 2006, vol. 11, no. 1, pp. 58–61. Publisher Full Text\n\nDietz C, et al.: Integration of the ImageJ Ecosystem in KNIME Analytics Platform. Front. Comput. Sci. Mar. 2020; 2(8), p. 8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPietzsch T, Preibisch S, Tomančák P, et al.: ImgLib2—generic image processing in Java. Bioinformatics. Nov. 2012; 28(22): 3009–3011. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHong NC: In which journals should I publish my software?Reference Source\n\nGiving software its due. Nat. Methods. 01-Mar-2019; 16(3). Nature Publishing Group, p. 207. PubMed Abstract \| Publisher Full Text\n\nCall for Proposal ‘Research Software Sustainability,’.2016. Reference Source\n\nCZI Announces Support for Open-Source Software Efforts to Improve Biomedical Imaging.2018. Reference Source" }	[ { "id": "83552", "date": "04 May 2021", "name": "Loïc A. Royer", "expertise": [ "Reviewer Expertise bioimage analysis", "microscopy", "biology", "algorithms", "optics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe opinion article \"Developing open-source software for bioimage analysis: opportunities and challenge.\" addresses the important topic of developing software for bioimage analysis.\n\nThe authors discuss all aspects including the genesis of a project, the choice of scope and audience, technical choices, the role of communities, the ever important documentation, licensing, publication, and the crucial aspect of long-term funding and maintenance.\nI wholeheartedly agree with everything said here. Nothing here is controversial or in my opinion subject to debate. The issue perhaps is that the bits of wisdom in this piece should be more broadly shared, and in that sense I welcome its publication. If debate must arise, let the community as a whole do the debating.\nHere comes some constructive feedback to improve the manuscript: I find the first figure a bit cryptic, the different symbols are nice but there is no legend, and they are combined in evocative but unclear ways. It takes a bit too long to 'decode' the figure in my honest opinion. I would rethink the graphical design, make things more explicit, and add a bit of text to guide the reader.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] }, { "id": "87725", "date": "24 Jun 2021", "name": "Nico Stuurman", "expertise": [ "Reviewer Expertise Cell Biology", "single molecule imaging", "open source software for microscope control." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript, written by leaders in the field, provides a wonderful overview of the considerations, pitfalls, and challenges for developers of open source software for bioimage analysis. It is of interest to users, who will get a taste of the efforts behind the tools they are using, and to (aspiring) developers, who are provided with a great overview of all aspects of tool development, helping them make sound choices in their work. The manuscript is well organized, well written, and frankly, a pleasure to read. There are a few more subjects/questions related to bioimage analysis software development that I am interested in, and would love to see the authors’ take:\nThe relation to commercial software. Back a few decades ago, algorithms were developed in academia, and User Interfaces to work with these algorithms were created by commercial companies (NIH Image / ImageJ was one of the first software packages to break this “tradition”, and received significant push back from commercial entities). I would love to see a description of the current relation between open source and commercial bioimaging software, as well as a description what this relation ideally would be.\n\nPerhaps related and perhaps partly covered: the tension between creating new approaches to problems for which solutions exist (open source or not), versus using/embracing/extending existing solutions. The software development world in general has adopted the strategy to try many approaches and let the most widely used or durable solution(s) stand. Due to the limited resources in the bioimage software world, this hardly seems the most optimal approach, yet many are tempted to make a better solution than the one that already exists.\n\nDistribution: Code signing / security. Operating systems and IT infrastructures are increasing security requirements for desktop software, and a future where unsigned/non-confirming software can no longer be executed seems near, yet there are few (I don’t remember encountering any) one click installers of bioimage analysis software that are signed, ImageJ needs moving the executable out of and back into the Applications folder to get it to run on modern OSX, and Fiji advices against installing into the standard “Program Files” directory on Windows. I am not sure whether this is a minor financial and technical hurdle to be addressed by individual developers or something that needs more community attention.\n\nAgain, the manuscript is great as is, and would totally understand if the authors feel these issues are out of scope.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-302
https://f1000research.com/articles/10-301/v1	19 Apr 21	{ "type": "Systematic Review", "title": "Global prevalence of prolonged gastrointestinal symptoms in COVID-19 survivors and potential pathogenesis: A systematic review and meta-analysis", "authors": [ "Fauzi Yusuf", "Marhami Fahriani", "Sukamto S. Mamada", "Andri Frediansyah", "Azzaki Abubakar", "Desi Maghfirah", "Jonny Karunia Fajar", "Helnida Anggun Maliga", "Muhammad Ilmawan", "Talha Bin Emran", "Youdiil Ophinni", "Meutia Rizki Innayah", "Sri Masyeni", "Abdulla Salem Bin Ghouth", "Hanifah Yusuf", "Kuldeep Dhama", "Firzan Nainu", "Harapan Harapan", "Marhami Fahriani", "Sukamto S. Mamada", "Andri Frediansyah", "Azzaki Abubakar", "Desi Maghfirah", "Jonny Karunia Fajar", "Helnida Anggun Maliga", "Muhammad Ilmawan", "Talha Bin Emran", "Youdiil Ophinni", "Meutia Rizki Innayah", "Sri Masyeni", "Abdulla Salem Bin Ghouth", "Hanifah Yusuf", "Kuldeep Dhama", "Firzan Nainu", "Harapan Harapan" ], "abstract": "Background: This study aimed to determine the cumulative prevalence of prolonged gastrointestinal (GI) symptoms, including nausea, vomiting, diarrhea, lack of appetite, abdominal pain, and dysgeusia, in survivors of both mild and severe COVID-19 worldwide and to discuss the potential pathogenesis.\n\nMethods: Three databases (PubMed, Scopus, and Web of Science) were searched for relevant articles up to January 30, 2021. Data on study characteristics, clinical characteristics during follow-up, the number of patients with prolonged GI symptoms, and total number of COVID-19 survivors were retrieved according to PRISMA guidelines. The quality of eligible studies was assessed using the Newcastle-Ottawa scale. The pooled prevalence of specific prolonged GI symptoms was calculated and the association between COVID-19 severity and the occurrence of prolonged GI symptoms was assessed if appropriate.\n\nResults: The global prevalence of prolonged nausea was 3.23% (95% CI: 0.54%–16.53%) among 527 COVID-19 survivors. Vomiting persisted in 93 of 2,238 COVID-19 survivors (3.19%, 95% CI: 1.62%–6.17%) and prolonged diarrhea was found in 34 of 1,073 survivors (4.12%, 95% CI: 1.07%–14.64%). A total of 156 patients among 2,238 COVID-19 survivors (4.41%, 95% CI: 1.91%–9.94%) complained of persistent decreased or loss of appetite. The cumulative prevalence of prolonged abdominal pain was 1.68% (95% CI: 0.84%–3.32%), whereas persistent dysgeusia was identified in 130 cases among 1,887 COVID-19 survivors (7.04%, 95% CI: 5.96%–8.30%). Data was insufficient to assess the relationship between COVID-19 severity and the occurrence of all prolonged GI symptoms.\n\nConclusion: Persistent GI symptoms among COVID-19 survivors after discharge or recovery raises a concern regarding the long-term impact of the COVID-19 infection on the quality of life of the survivors. Despite several potential explanations proposed, studies that aim to follow patients after recovery from COVID-19 and determine the pathogenesis of the prolonged symptoms of COVID-19 survivors are warranted.\n\nPROSPERO registration: CRD42021239187.", "keywords": [ "COVID-19", "prolonged symptom", "long-term effect", "gastrointestinal", "systematic review" ], "content": "Introduction\n\nThe coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was initially confirmed in late December 2019 in Wuhan, China, and spread quickly globally and has become a global pandemic. As of February 6, 2021, over one hundred million confirmed cases worldwide and more than 2.5 million deaths have been reported.1 COVID-19 has affected both the healthcare system2–5 and socioeconomics6,7 across the globe. Numerous treatment-related drug proposals8–10 and vaccine development programs11,12 for COVID-19 continue to be investigated, despite the many unknowns. There are no official drugs for COVID-19 that are recommended by the World Health Organization (WHO) with some treatments recommended solely on the basis of clinical trials.\n\nThe SARS-CoV-2 infection mainly affects the respiratory system, however, various other organs can also be affected13–17 and with many unknown outcomes. Several studies have been conducted to assess the effects of SARS-CoV-2 on several affected health outcomes including those of the hepatic,18 cardiovascular,19,20 and central nervous systems,21,22 and the occurrence of anosmia and dysgeusia,23 as well as hemorrhagic and ischemic stroke.24 Recently, gastrointestinal (GI) problems have emerged in patients with COVID-19, in particular diarrhea.25 SARS-CoV-2 has been found in infected feces26,27 and contaminated water supply.28,29 A study reported the detection of the SARS-CoV-2 in the stool of 54% of infected patients.30 The first connection of COVID-19 with GI problems was established in patients with COVID-19 in Wuhan, Hubei Province, China.31 Patients with GI problems were required to stay at the hospital longer than those without GI problems.31 New cases of GI symptoms have also been found in western countries. Cohort reports from the USA showed that approximately 60% of 318 patients had GI symptoms.32 In the United Kingdom, a report showed that eight children with COVID-19 had atypical appendicitis symptoms.33\n\nRecent evidence suggests that the GI symptoms in patients with COVID-19 could be persistent.34,35 A study in the USA found that 87.4% of patients who had recovered from COVID-19 reported persistence of at least one symptom including GI symptoms.34 However, the magnitude of this persistent or prolonged occurrence of GI symptoms in those who have recovered from COVID-19 (survivors) is missing in the literature. The pathogenesis mechanisms of prolonged GI symptoms in SARS-CoV-2 infection are also scarce. In general, GI problems are accompanied by intestinal damage or inflammation.36 The loss of barrier integrity in the intestine results in the invasion of microbes that could induce adaptive and immune cells, including dendritic cells.37,38 However, the pathogenesis of GI problems in COVID-19 needs to be elucidated to inform better prevention and treatment approaches. The objective of this systematic review and meta-analysis was (a) to determine the global prevalence of prolonged GI symptoms including nausea, vomiting, diarrhea, lack of appetite, abdominal pain, and dysgeusia in those who had recovered from mild and severe COVID-19 and (b) to determine the association of COVID-19 severity with prolonged GI symptoms. In addition, the potential pathogenesis of these GI symptoms is also discussed.\n\n\nMethods\n\nThe protocol of this study was registered in PROSPERO (CRD42021239187) and the protocol required no ethical clearance. To ensure the robustness of the generated data, we followed the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines to search electronic databases and report our findings.39 The completed PRISMA checklist is presented in Figshare.40\n\nStudies reporting at least one prolonged or persistent GI symptom such as nausea, vomiting, diarrhea, lack or loss of appetite, abdominal pain, and dysgeusia in patients with COVID-19 after being discharged from hospital were considered eligible. Editorials, commentaries, reviews, case reports, and case series were excluded. Diagnosis of COVID-19 must have been confirmed using RT-PCR of SARS-CoV-2 RNA from nasal or oropharyngeal swab samples. Studies that diagnosed patients with COVID-19 based on symptoms only (without nucleic acid testing) were excluded. COVID-19 survivors were defined as all patients with COVID-19 who met either the WHO or China National Health Commission discharge criteria.41,42 Prolonged GI symptoms were defined as persistence of symptoms for at least two weeks after discharge in COVID-19 survivors.\n\nThe potential articles in three databases (PubMed, Scopus, and Web of Science) were searched as of January 30, 2021. The searches were limited to 2019-2021 and only articles written in English were considered eligible. The search strategies were as follows. PubMed ([Title](“SARS-CoV-2” OR “COVID-19” OR “Wuhan coronavirus” OR “Wuhan virus” OR “novel coronavirus” OR “nCoV” OR “severe acute respiratory syndrome coronavirus 2” OR “coronavirus disease 2019” OR “2019-nCoV” OR “2019 novel coronavirus” OR “SARS 2”) AND ([Title](“prolong” OR “follow-up” OR “persistent” OR “sequelae” OR “consequen” OR “prospective” OR “cohort” OR “long-term” OR “follow” OR “longitudinal”). Web of Science ([Title](“SARS-CoV-2” OR “COVID-19” OR “Wuhan coronavirus” OR “Wuhan virus” OR “novel coronavirus” OR “nCoV” OR “severe acute respiratory syndrome coronavirus 2” OR “coronavirus disease 2019” OR “2019-nCoV” OR “2019 novel coronavirus” OR “SARS 2”) AND ([Title](“prolong” OR “follow-up” OR “persistent” OR “sequelae” OR “consequen” OR “prospective” OR “cohort” OR “long-term” OR “follow” OR “longitudinal”). Scopus ([Title](“SARS-CoV-2” OR “COVID-19” OR “Wuhan coronavirus” OR “Wuhan virus” OR “novel coronavirus” OR “nCoV” OR “severe acute respiratory syndrome coronavirus 2” OR “coronavirus disease 2019” OR “2019-nCoV” OR “2019 novel coronavirus” OR “SARS 2”) AND ([Title](“prolong” OR “follow-up” OR “persistent” OR “sequelae” OR “consequen” OR “prospective” OR “cohort” OR “long-term” OR “follow*” OR “longitudinal”).\n\nEssential information of all articles was imported to a reference manager (EndNote X9, Thompson Reuters, Philadelphia, PA, USA) and duplicated records among the three databases were removed. The titles and abstracts of all records were screened to identify eligible articles. The full texts of potentially eligible studies were downloaded and reviewed by two authors (MF and HH). The eligibility of each study was decided based on the eligibility criteria and the availability of the data. Data extraction was conducted as explained in previous studies.23,24,43 Briefly, important data from the eligible articles were extracted and whenever required supplementary materials were extracted. The list of references was retrieved to search for additional relevant studies. The collected study characteristics of the eligible articles included author(s), year of study, study site and country, study design, extent of follow-up conducted after discharge, number of patients with COVID-19, number of patients with COVID-19 with prolonged specific GI symptoms, and severity of the COVID-19 infection during admission to the hospital.\n\nTwo main outcomes were evaluated in this study: (a) global prevalence of prolonged GI symptoms including nausea, vomiting, diarrhea, lack of appetite, abdominal pain, and dysgeusia and (b) association of COVID-19 severity with the presence of prolonged GI symptoms (nausea, vomiting, diarrhea, lack of appetite, abdominal pain, and dysgeusia). In addition, the possible pathogenesis mechanisms of GI symptoms in COVID-19 including those with prolonged GI symptoms are discussed.\n\nThe prevalence of each prolonged GI symptom (nausea, vomiting, diarrhea, lack of appetite, abdominal pain, and dysgeusia) was calculated as the number of patients with a prolonged symptom divided by the total number of patients with COVID-19 with or without the specific GI symptom during the follow-up and expressed as frequency (%) with a 95% confidence interval (CI). The associations of COVID-19 severity and the risk of prolonged GI symptoms were also calculated. Forest plots were used to visualize the data.\n\nThe Newcastle-Ottawa scale (NOS) was used for critical assessment of the quality of each included study.44 The NOS evaluates nine characteristics of a study including four, one, and three items for sample selection, group comparison, and the outcome, respectively. The scores range between 0 to 9 and a study is classified into one of three groups based on the score: low (≤ 4), moderate (between 5–6), and high-quality (≥ 7) study.\n\nThe Q test was used to evaluate the heterogeneity of the pooled data and the data was analyzed using a random-effect or fixed-effect model as appropriate. Egger’s test was used to assess publication bias (a p < 0.05 is considered indicative of potential publication bias). The associations between severity of COVID-19 and the risk of GI symptoms were calculated using the Z test. Review Manager version 5.3 was used to analyze the data.45\n\n\nResults\n\nThe database searches yielded 4,050 eligible original articles, with 2,005 publications remaining after the duplicates were removed. Initial screening of the titles and abstract excluded 1,244 articles, leaving 761 studies (Figure 1). After reviewing the full-texts of these studies, an additional 739 articles were excluded for several reasons including that they were reviews, case series, case reports, initial reports on COVID-19, letters or commentaries, studies on specific groups, recommendations, clinical trials, and studies with insufficient data. The final screening resulted in 22 articles which were included in this meta-analysis.\n\nAmong the 22 studies selected, the meta-analysis to calculate the prevalence of prolonged GI symptoms included four studies for nausea,35,46–48 three studies for vomiting,35,49,50 seven studies for diarrhea,35,46–48,50–52 three studies for loss of or decrease in appetite,35,49,50 three studies for abdominal pain,46,47,50 and six studies for dysgeusia.46,47,49,53–55 The studies included for the prevalence of prolonged GI symptoms are summarized in Table 1.\n\nThe information about COVID-19 severity on admission and the occurrence of GI symptoms were provided in one article each for nausea35 and dysgeusia,49 and three articles each for vomiting35,49,50 and loss of or decrease in appetite.35,49,50\n\nProlonged nausea was reported in 10 patients after recovery, with estimated prevalence of 3.23% (95% CI: 0.54%–16.53%) from a total of 527 patients with COVID-19 from four studies (Figure 2). Persistent vomiting was identified in 93 of 2,238 patients with COVID-19 from three studies, which corresponded to a pooled prevalence of 3.19% (95% CI: 1.62%–6.17%). Seven articles reported the prevalence of prolonged diarrhea as 4.12% (34/1,073 patients with COVID-19) with 95% CI: 1.07%–14.64%. Loss of or decrease in appetite was reported in three studies that included 2,238 patients with COVID-19, among whom 156 patients were reported to have had the symptom (estimated prevalence of 4.41%, 95% CI: 1.91%–9.94%). Based on three studies, abdominal pain was reported in 8/511 patients with COVID-19 with an estimated prevalence of 1.68% (95% CI: 0.84%–3.32%). Six studies identified 130 cases of prolonged dysgeusia among a total of 1,887 patients with COVID-19 (7.04%, 95% CI: 5.96%–8.30%). Abdominal pain and dysgeusia were analyzed using a fixed-effect model of Egger’s test at p < 0.001.\n\n(A) Estimated prevalence of prolonged nausea in COVID-19 (event rate 3.23%, 95%CI: 0.54%–16.53%, p < 0.001, p Egger 1.683, and p heterogeneity <0.001). (B) Estimated prevalence of prolonged vomiting in COVID-19 patients (event rate 3.19%, 95%CI: 1.62%–6.17%, p = 0.028, p Egger 0.482, and p heterogeneity 0.028. (C) Estimated prevalence of prolonged diarrhea in COVID-19 patients (event rate 4.12%, 95%CI: 1.07%–14.64%, p < 0.001, p Egger 1.726, and p heterogeneity <0.001). (D) Estimated prevalence of prolonged of loss of appetite in COVID-10 patients (event rate 4.41%, 95%CI: 1.91%–9.94%, p < 0.001, p Egger 0.690, and p heterogeneity <0.001. (E) Estimated prevalence of prolonged of abdominal pain in COVID-19 patients (event rate 1.68%, 95%CI: 0.84%–3.32%, p = 0.499, p Egger <0.001, and p heterogeneity 0.499). (F) Estimated prevalence of prolonged of dysgeusia in COVID-19 patients (event rate 7.04%, 95%CI: 5.96%–8.30%, p = 0.526, p Egger <0.0001, and p heterogeneity 0.052).\n\nOwing to the lack of studies presenting prolonged GI symptoms among patients with mild-moderate and severe COVID-19, the associations were calculated only for vomiting and loss of appetite. The severity of COVID-19 was not associated with the presence of either vomiting or loss of appetite in patients with COVID-19 (odds ratio (OR): 1.19, 95% CI: 0.51–2.78 and OR: 0.84, 95% CI: 0.47–1.5, respectively). Both symptoms were analyzed using the fixed-effect model of Egger’s test at p < 0.001.\n\nStudies have confirmed that the existence of SARS-CoV-2 in the GI tract can last for several weeks after a throat swab shows a negative result.56,57 One study found that the elimination of SARS-CoV-2 from fecal samples was completed more than a month after samples collected from the respiratory tract turned out negative.58 This may explain the prolonged GI symptoms observed in patients with COVID-19 in the present study.\n\nTo date, no satisfying explanation is available as to why the virus lasts longer in the gut than in the other systems. Although the exact mechanisms are not fully elucidated, some putative pathophysiological mechanisms underlying the occurrence of COVID-19-induced GI symptoms have been put forward. These potential mechanisms encompass the direct invasion of SARS-CoV-2 in GI cells, secondary effects after other organs are infected, and drug treatment-induced digestive symptoms.59,60\n\nIt has been shown that angiotensin-converting enzyme 2 (ACE2), the entry receptor of SARS-CoV-2, is also highly expressed in the digestive system organs such as the esophagus, small intestine, and colon.61,62 Therefore, it is quite plausible to observe the advent of several GI symptoms induced by SARS-CoV-2 infection in patients ranging from nausea, vomiting, and diarrhea to loss of appetite and abdominal pain.59,60,63 The expression of the ACE2 receptor in the gut results in the digestive system also being vulnerable to attack by SARS-CoV-2. After being occupied by the virus, ACE2 becomes dysfunctional, resulting in the impairment of the protective activity of the ACE2/Ang-(1–7)/Mas axis, whereas the activity of the ACE/Ang II/AT1R axis is elevated.64 Following this condition, nicotinamide adenine dinucleotide phosphate oxidases are excessively activated leading to the occurrence of oxidative stress-induced inflammation and this finally causes tissue damage as described in Figure 3.65,66\n\nThe inactive Ang I is converted into Ang II which produces its biological activities via its binding to AT1R. To maintain the homeostasis status, the catalytic activity of ACE2 converts Ang II to Ang 1-7 which has the opposite action of Ang II through its binding to Mas receptor. The invasion of SARS-CoV-2 to ACE2 causes an accumulation of Ang II and decreased level of Ang 1-7. This condition is linked to the increased interaction between Ang II and its receptor AT1R resulting in the hyperactivity of NADPH oxidase which is related to oxidative stress, massive production of proinflammatory cytokines, increased activity of thrombogenic factors and vasoconstriction. These events are eventually associated with the emergence of the COVID-19 GIT symptoms.\n\nFurthermore, when the virus invades the digestive system, the immune cells move to the site of infection and release a massive amount of proinflammatory cytokines, such as interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-alpha (TNF-α) resulting in intestinal inflammation.67 The inflammation of the intestines was confirmed in a study which found that the level of fecal calprotectin, a specific protein biomarker for intestinal inflammation found in the feces, increased in patients with COVID-19.68 Interestingly, the rise in calprotectin is higher in patients who are also suffering from diarrhea68 indicating that diarrhea in SARS-CoV-2 infection might be linked to intestinal inflammation. It has been proposed that intestinal inflammation might cause diarrhea by disturbing the homeostasis of gut microbiota.69 Indeed, several inflammation-related diseases such as ulcerative colitis70 and Crohn’s disease71 are treated using probiotics to overcome gut dysbiosis.\n\nDiarrhea observed in patients with COVID-19 can also be linked to the impairment of the noncatalytic activity of ACE2. The receptor plays a pivotal role in the uptake of neutral amino acids such as tryptophan.72 The uptake of tryptophan into the enterocytes depends on the activity of the B0AT1 transporter which is colocalized with ACE2 to act properly.73 Thus, any dysfunctionalities in ACE2 will perturb the uptake of tryptophan by the cells. Moreover, the disturbances in tryptophan uptake are associated with the decreased activity of the mammalian target of rapamycin (mTOR) signaling pathway which has a responsibility in regulating the expression of antimicrobial peptides secreted by various intestinal cells and this will finally disturb the homeostasis of the gut microbiota.72–75 By this action, SARS-CoV-2 can induce GI symptoms, such as diarrhea, as observed in patients with COVID-19.76\n\nFurthermore, the suppression of the intestinal commensal microbes can result in worse consequences as these microbes are significantly involved in the regulation of microbiota homeostasis.77 Specifically, in addition to the intestinal cells, antimicrobial peptides, such as short-chain fatty acids (acetate, butyrate, and propionate), can also be produced by the commensals.77 These fatty acids can activate G-protein coupled receptors found in the apical area of the intestinal cells, such as GPR43.78 GPR43 activation is followed by the induction of the mammalian target of rapamycin signaling pathway which has previously been described as the pathway responsible for regulating the expression of antimicrobial peptides, such as defensins and RegIIIγ.79\n\nGut microbiomes are also implicated in immune responses where they are found to inhibit the action of proinflammatory cytokines such as IL-1β, IL-6, and TNF-α and to promote the action of anti-inflammatory cytokines such as IL-10.77,78 Therefore, the imbalance in microbiomes homeostasis could lead to the exacerbation of intestinal inflammation. Another critical role played by the intestinal flora is associated with the regulation of barrier integrity of the intestinal epithelia as it has been found that the flora is involved in the upregulation of tight junction proteins and promotion of mucus secretion.77 Taken together, the perturbation of the intestinal flora homeostasis may induce gut inflammation and promote GI symptoms as observed in patients with COVID-19 (Figure 4).\n\nThe neutral amino acid, such as tryptophan, is taken up by the intestinal cells through the action of an influx transporter B0AT1. To act properly, this transporter works together with ACE2. The activity of ACE2 is independent of RAS system. Once absorbed, tryptophan activates mTOR signaling pathway responsible for the regulation of intestinal antimicrobial peptides expression. During the COVID-19 course, ACE2 is invaded by SARS-CoV-2 disturbing the uptake of the amino acids by B0AT1. This condition is then followed by the inhibition of mTOR pathway resulting in the perturbances of antimicrobial peptides (i.e. defensins and RegIIIγ) secretion into the intestinal lumen. Furthermore, dysbiosis can cause several subsequent effects because it induces the production of proinflammatory cytokines, inhibits anti-inflammatory cytokines, weakens the tightness of the epithelial barrier and decreases the secretion of some beneficial metabolites from the microbiomes. Taken together, these effects result in the emergence of GIT symptoms, such as intestinal inflammation and diarrhea.\n\nThe steady-state of gut microbiota could also be impaired by changes in oxygen supply in the intestine as hypoxia is seen in a large portion of patients with COVID-19.80,81 It has been reported that the microbiome has a critical role in maintaining oxygen levels in the gut to promote the absorption of various nutrients, regulation of the epithelial barrier, and response of the immune system.81\n\nThe interrelation between the digestive system and other systems or organs inspires another proposed mechanism underlying the emergence of GI symptoms during COVID-19 infection (Figure 5). As SARS-CoV-2 is not always detected in the feces of patients with COVID-19 who are also displaying GI symptoms, a study speculated that the symptoms are not usually linked to the direct invasion of the virus into the intestines.59 For example, through the gut-lung axis, dysbiosis in the gut is putatively linked to the disturbances in the respiratory flora and vice versa.59,82,83 It has been reported that the level of lung-derived C-C chemokine receptor type 9 (CCR9), a chemokine receptor required by CD4+ to move to the small intestine, increases during respiratory influenza virus infection.84 The movement of CCR9-CD4+ T cells to the intestine is promoted by the high abundant expression of CCL25 in the small intestine.59,85 CCL25/CCR9 is found to have a critical responsibility in directing the recruitment of lymphocytes to the small intestine which is subsequently followed by the disruption of intestinal flora homeostasis.59,86\n\nGIT symptoms during the COVID-19 course can be influenced by the virus infections in several sites such as in the CNS, lung and liver. The infections occurring in those organs can be sensed by the GIT leading to the emergence of the symptoms. Several axes are involved in this interrelationship such as the gut-brain axis which is mainly mediated by the enteric nervous system, gut-lung axis involving the movement of CCR9-CD4+ from the lung to the intestine driven by the CCL25, and gut-liver axis which is connected through the portal vein and biliary tract.\n\nSeveral studies suggest that the central nervous system could also be affected by SARS-CoV-2 in addition to the respiratory system as the main system invaded by the virus.87,88 The infection in the CNS might also affect the digestive system as patients with COVID-19 display some neurological-related GI symptoms, such as nausea and vomiting, without SARS-CoV-2 detection in their stool.59,89 Conversely, the gut-brain axis might also provide another entry route for the virus to reach the brain.89 The gut-brain axis is connected by the enteric nervous system which is a unique autonomous nervous system because it has both sensory and motor properties.90 Although most of its neurons are not directly innervated by the CNS, critical reciprocal communication occurs between the CNS and the GI system through the enteric nervous system.90 Thus, there is speculation that the involvement of the enteric nervous system as a bridge allows bidirectional passage of either the virus or proinflammatory cytokines.83\n\nFinally, the gut-liver axis must not be overlooked. The invasion and replication of SARS-CoV-2 in the intestine can weaken the epithelial barrier and cause leakage in the gut-blood barrier resulting in the spread of either the virus or its metabolites systemically.83 Junctional proteins and the extracellular matrix as critical components of most barriers in the body, including the gut-blood barrier,91 could be impaired by proinflammatory cytokines.66,92–95 The weakening of the barrier could also pave the way for the intestinal flora to reach the liver via the portal vein.59 In turn, through the biliary tract, the liver, supported by cholangiocytes, could transfer microbial metabolites and cytokines into the gut system59,96 which may eventually initiate the GI symptom. Interestingly, one study speculates that retrograde movement of the virus to the liver through the biliary tract should also be taken into account.97\n\nThe various drugs administered during the COVID-19 course could be linked to the emergence of GI symptoms as adverse reactions. As shown in antimicrobial agents, antiviral agents can also change the steady-state level of the gut microbiota which can cause diarrhea.59 Some antibiotics, such as cephalosporins, penicillins, quinolones, and macrolides are known to induce diarrhea when they are used to treat infections.59,98 During COVID-19 infection, the use of these antibiotics is common, and has been correlated with the increased number of drug-induced diarrhea cases.59,99,100 In general, pharmacological agents can cause diarrhea via a number of mechanisms such as by disturbing the normal flora that reside in the gut, promoting the growth of pathogenic microbes, inducing allergic or toxic reactions in the intestinal mucosa, or by stimulating the motility of the gut.101 In particular, the use of broad-spectrum antibiotics such as penicillins and cephalosporins, is found to be one of the causes of Clostridium difficile hegemony over the normal intestinal microbiota.102,103 This occurs as the antimicrobial agents could kill the flora while leaving the pathogenic microorganisms without control from the normal flora.101\n\nSeveral antiviral agents are also reported to have GI symptoms as their adverse effects when administered to patients with COVID-19. The use of remdesivir, lopinavir, and ritonavir was found to induce nausea and vomiting.104 The increased level of noxious chemicals, including drugs, in the GI tract could send a signal to the vomiting center in the CNS through afferent fibers of the glossopharyngeal and vagal nerves to induce emesis.105\n\n\nConclusion\n\nAlthough the pooled prevalence of prolonged GI symptoms in COVID-19 survivors is low, this study adds new insights to the long-term impact of COVID-19 in recovered patients. This systematic review will help increase awareness among clinicians regarding potentially prolonged consequences of COVID-19. Follow-up cohort studies should be designed and managed to identify the effect of this pandemic on the quality of life of the survivors.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nFigshare: PRISMA checklist for ‘Global prevalence of prolonged gastrointestinal symptoms in COVID-19 survivors and potential pathogenesis: A systematic review and meta-analysis’, https://doi.org/10.6084/m9.figshare.14083613.106\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC BY 4.0).", "appendix": "Acknowledgement\n\nAuthors would like to thank Narra Studio Jurnal Indonesia in assisting during writing processes.\n\n\nReferences\n\nWorldometers: COVID-19 coronavirus pandemic.Reference Source [cited 2021 February 6].\n\nGreene CJ, Burleson SL, Crosby JC, et al.: Coronavirus disease 2019: International public health considerations. 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Microbiol Spectr. 2017; 5(5). eng. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nParada Venegas D, De la Fuente MK, Landskron G, et al.: Short Chain Fatty Acids (SCFAs)-Mediated Gut Epithelial and Immune Regulation and Its Relevance for Inflammatory Bowel Diseases. Front Immunol. 2019 2019-March-11; 10(277). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhao Y, Chen F, Wu W, et al.: GPR43 mediates microbiota metabolite SCFA regulation of antimicrobial peptide expression in intestinal epithelial cells via activation of mTOR and STAT3. Mucosal Immunol. 2018 2018/05/01; 11(3): 752–62. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCavezzi A, Troiani E, Corrao S: COVID-19: hemoglobin, iron, and hypoxia beyond inflammation. A narrative review. Clin Pract. 2020 May 19; 10(2): 1271. Epub 2020/06/09. eng. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSinghal R, Shah YM: Oxygen battle in the gut: Hypoxia and hypoxia-inducible factors in metabolic and inflammatory responses in the intestine. J Biol Chem. 2020 Jul 24; 295(30): 10493–505. Epub 2020/06/07. eng. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKeely S, Talley NJ, Hansbro PM: Pulmonary-intestinal cross-talk in mucosal inflammatory disease. Mucosal Immunol. 2012 Jan; 5(1): 7–18. Epub 2011/11/18. eng. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTrottein F, Sokol H: Potential Causes and Consequences of Gastrointestinal Disorders during a SARS-CoV-2 Infection. Cell Rep. 2020 Jul 21; 32(3): 107915. Epub 2020/07/11. eng. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang J, Li F, Wei H, et al.: Respiratory influenza virus infection induces intestinal immune injury via microbiota-mediated Th17 cell–dependent inflammation. J Exp Med. 2014; 211(13): 2683. eng. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPapadakis KA, Prehn J, Nelson V, et al.: The role of thymus-expressed chemokine and its receptor CCR9 on lymphocytes in the regional specialization of the mucosal immune system. J Immunol. 2000 Nov 1; 165(9): 5069–76. Epub 2000/10/25. eng. PubMed Abstract \| Publisher Full Text\n\nSvensson M, Agace WW: Role of CCL25/CCR9 in immune homeostasis and disease. Expert Rev Clin Immunol. 2006 Sep; 2(5): 759–73. Epub 2006/09/01. eng. PubMed Abstract \| Publisher Full Text\n\nHelms J, Kremer S, Merdji H, et al.: Neurologic Features in Severe SARS-CoV-2 Infection.N Engl J Med.2020 Jun 4; 382(23): 2268–70. Epub 2020/04/16. eng. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPaniz-Mondolfi A, Bryce C, Grimes Z, et al.: Central nervous system involvement by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). J Med Virol. 2020 Jul; 92(7): 699–702. Epub 2020/04/22. eng. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBostancıklıoğlu M: Temporal Correlation Between Neurological and Gastrointestinal Symptoms of SARS-CoV-2. Inflamm Bowel Dis. 2020 Jul 17; 26(8): e89–e91. Epub 2020/05/23. eng. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRao M, Gershon MD: The bowel and beyond: the enteric nervous system in neurological disorders. Nat Rev Gastroenterol Hepatol. 2016 2016/09/01; 13(9): 517–28. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVancamelbeke M, Vermeire S: The intestinal barrier: a fundamental role in health and disease. Expert Rev Gastroenterol Hepatol. 2017; 11(9): 821–34. Epub 2017/06/26. eng. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMountain DJ, Singh M, Menon B, et al.: Interleukin-1beta increases expression and activity of matrix metalloproteinase-2 in cardiac microvascular endothelial cells: role of PKCalpha/beta1 and MAPKs. Am J Physiol Cell Physiol. 2007 Feb; 292(2): C867–75. 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PubMed Abstract \| Publisher Full Text\n\nPinto C, Giordano DM, Maroni L, et al.: Role of inflammation and proinflammatory cytokines in cholangiocyte pathophysiology. Biochim Biophys Acta Mol Basis Dis. 2018 2018/04/01/; 1864(4, Part B): 1270–8. PubMed Abstract \| Publisher Full Text\n\nGalanopoulos M, Gkeros F, Doukatas A, et al.: COVID-19 pandemic: Pathophysiology and manifestations from the gastrointestinal tract. World J Gastroenterol. 2020 Aug 21; 26(31): 4579–88. Epub 2020/09/05. eng. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDhar D, Mohanty A: Gut microbiota and Covid-19- possible link and implications. Virus Res. 2020 Aug; 285: 198018. Epub 2020/05/21. eng. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSultana J, Cutroneo PM, Crisafulli S, et al.: Azithromycin in COVID-19 Patients: Pharmacological Mechanism, Clinical Evidence and Prescribing Guidelines. Drug Saf. 2020; 43(8): 691–8. eng. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTanioka H, Tanioka S: Risks and Benefits of Antibiotics vs. COVID-19 Morbidity and Mortality. medRxiv. 2020: 2020.10.15.20213603. Publisher Full Text\n\nHögenauer C, Hammer HF, Krejs GJ, et al.: Mechanisms and management of antibiotic-associated diarrhea. Clin Infect Dis. 1998 Oct; 27(4): 702–10. Epub 1998/11/03. eng. PubMed Abstract \| Publisher Full Text\n\nKociolek LK, Gerding DN: Breakthroughs in the treatment and prevention of Clostridium difficile infection. Nat Rev Gastroenterol Hepatol. 2016 Mar; 13(3): 150–60. Epub 2016/02/11. eng. PubMed Abstract \| Publisher Full Text\n\nPothoulakis C: Pathogenesis of Clostridium difficile-associated diarrhoea. Eur J Gastroenterol Hepatol. 1996 Nov; 8(11): 1041–7. Epub 1996/11/01. eng. PubMed Abstract \| Publisher Full Text\n\nZhang T, Liu D, Tian D, et al.: The roles of nausea and vomiting in COVID-19: did we miss something? J Microbiol Immunol Infect. 2020. eng. Publisher Full Text \| Free Full Text\n\nBecker DE: Nausea, vomiting, and hiccups: a review of mechanisms and treatment. Anesth Prog. 2010 Winter; 57(4): 150–7. eng. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHarapan H, Yusuf FGlobal prevalence of prolonged gastrointestinal symptoms in COVID-19 survivors and potential pathogenesis - A systematic review and meta-analysis. figshare. Journal contribution. 2021. Publisher Full Text" }	[ { "id": "83534", "date": "05 May 2021", "name": "Ari Fahrial Syam", "expertise": [ "Reviewer Expertise Gastroenterology", "GERD", "H pylori", "GERD and COVID-19", "Endoscopy and COVID-19" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI think that this meta-analysis is well done with the PRISMA guidelines. The quality of eligible studies from this meta-analysis was assessed using the Newcastle-Ottawa scale. The protocol of this study was also registered in PROSPERO, an international prospective register for systemic review.\nThe topics about GI symptops in COVID-19 survivors are also interesting and need to be of concern to clinicians.\nThe 22 studies that were eventually used in this study can be used to draw conclusions about the prevalence of GI symptoms that appear. The pathophysiological explanation for the occurrence of symptoms also makes sense.\nI think this article will benefit for clinician and community.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [] }, { "id": "85353", "date": "24 May 2021", "name": "Mahir Gachabayov", "expertise": [ "Reviewer Expertise Clinical outcomes research and evidence synthesis" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis systematic review and meta-analysis is aimed at evaluating the prevalence of prolonged GI symptoms and their associations with adverse outcomes among patients with COVID-19. Moreover, the authors aimed to describe possible pathogenetic mechanisms behind such symptoms.\nThe concept and the research question make sense. The abstract reflects the core of the findings and is well-written.\n\nIntroduction is comprehensible and easy to read, describes the gap in the current literature. The objective is stated clearly.\nMethods are adequate. The report complies with the PRISMA guidelines. The protocol of this systematic review was a priori registered in PROSPERO, a fact that mitigates the risks of reporting bias. Eligibility criteria were predefined and make sense. The term 'prolonged symptoms' was defined clearly, a fact that mitigates the risks of detection bias. Endpoints were defined clearly and are clinically relevant. Data sources and search strategy used were comprehensive and the details were reported, which makes the search reproducible. Quality assessment and statistical analysis were comprehensive.\nResults are well-written. PRISMA flow diagram shows nicely the flow of screening and study selection. Table 1 nicely summarizes included studies and Figure 2 nicely depicts the findings of the statistical analysis in forest plots.\nDiscussion is comprehensible and easy to read. The findings of this study were discussed in the context of the current evidence. Pathogenesis of the impact of SARS-CoV-2 on the GI tract was well-described alluding to the relevant literature.\nThe Conclusion is justified by the findings.\nI have a few comments:\nI think, the term 'COVID-19 patients' rather than \"COVID-19 survivors' should be used. The fact of the matter is that GI symptoms are a part of COVID-19 symptoms. In addition, mortality of GI symptoms was not evaluated to be 0%. Otherwise, the term 'COVID-19 respiratory disease survivors' may be used.\n\nDid the included studies discriminate and compare those who primarily had respiratory symptoms and those who had isolated GI disease? Please comment in the Discussion.\n\nI compliment the authors for recognizing and commenting on drug-induced GI symptoms. In fact, this entity may have confounded with the statistical findings. And in fact, this could be a reason behind considerable heterogeneity in primary endpoints.\n\nPlease add a brief paragraph to the end of the Discussion acknowledging the strengths and limitations of the systematic review.\n\nTable 1, Study Design: Please change the term 'Cohort' to 'Retrospective' to avoid confusion as prospective observational studies are also cohort studies.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-301
https://f1000research.com/articles/10-33/v1	18 Jan 21	{ "type": "Method Article", "title": "Sustainable data analysis with Snakemake", "authors": [ "Felix Mölder", "Kim Philipp Jablonski", "Brice Letcher", "Michael B. Hall", "Christopher H. Tomkins-Tinch", "Vanessa Sochat", "Jan Forster", "Soohyun Lee", "Sven O. Twardziok", "Alexander Kanitz", "Andreas Wilm", "Manuel Holtgrewe", "Sven Rahmann", "Sven Nahnsen", "Johannes Köster", "Felix Mölder", "Kim Philipp Jablonski", "Brice Letcher", "Michael B. Hall", "Christopher H. Tomkins-Tinch", "Vanessa Sochat", "Jan Forster", "Soohyun Lee", "Sven O. Twardziok", "Alexander Kanitz", "Andreas Wilm", "Manuel Holtgrewe", "Sven Rahmann", "Sven Nahnsen" ], "abstract": "Data analysis often entails a multitude of heterogeneous steps, from the application of various command line tools to the usage of scripting languages like R or Python for the generation of plots and tables. It is widely recognized that data analyses should ideally be conducted in a reproducible way. Reproducibility enables technical validation and regeneration of results on the original or even new data. However, reproducibility alone is by no means sufficient to deliver an analysis that is of lasting impact (i.e., sustainable) for the field, or even just one research group. We postulate that it is equally important to ensure adaptability and transparency. The former describes the ability to modify the analysis to answer extended or slightly different research questions. The latter describes the ability to understand the analysis in order to judge whether it is not only technically, but methodologically valid. Here, we analyze the properties needed for a data analysis to become reproducible, adaptable, and transparent. We show how the popular workflow management system Snakemake can be used to guarantee this, and how it enables an ergonomic, combined, unified representation of all steps involved in data analysis, ranging from raw data processing, to quality control and fine-grained, interactive exploration and plotting of final results.", "keywords": [ "data analysis", "workflow management", "sustainability", "reproducibility", "transparency", "adaptability", "scalability" ], "content": "1 Introduction\n\nDespite the ubiquity of data analysis across scientific disciplines, it is a challenge to ensure in silico reproducibility1–3. By automating the analysis process, workflow management systems can help to achieve such reproducibility. Consequently, a “Cambrian explosion” of diverse scientific workflow management systems is in process; some are already in use by many and evolving, and countless others are emerging and being published (see https://github.com/pditommaso/awesome-pipeline). Existing systems can be partitioned into five niches which we will describe below, with highlighted examples of each.\n\nFirst, workflow management systems like Galaxy4 and KNIME5 offer graphical user interfaces for composition and execution of workflows. The obvious advantage is the shallow learning curve, making such systems accessible for everybody, without the need for programming skills.\n\nSecond, with systems like Anduril6, Balsam7, Hyperloom8, Jug9, Pwrake10, Ruffus11, SciPipe12, SCOOP13, and COMPSs14 workflows are specified using a set of classes and functions for generic programming languages like Python, Scala, and others. Such systems have the advantage that they can be used without a graphical interface (e.g. in a server environment), and that workflows can be straightforwardly managed with version control systems like Git (https://git-scm.com).\n\nThird, with systems like Nextflow15, Snakemake16, BioQueue17, Bpipe18, ClusterFlow19, Cylc20, and BigDataScript21, workflows are specified using a domain specific language (DSL). Here, the advantages of the second niche are shared, while adding the additional benefit of improved readability; the DSL provides statements and declarations that specifically model central components of workflow management, thereby obviating superfluous operators or boilerplate code. For Nextflow and Snakemake, since the DSL is implemented as an extension to a generic programming language (Groovy and Python), access to the full power of the underlying programming language is maintained (e.g. for implementing conditional execution and handling configuration).\n\nFourth, with systems like Popper22, workflow specification happens in a purely declarative way, via configuration file formats like YAML23. These declarative systems share the concision and clarity of the third niche. In addition, workflow specification can be particularly readable for non-developers. The caveat of these benefits is that by disallowing imperative or functional programming, these workflow systems can be more restrictive in the processes that can expressed.\n\nFifth, there are system-independent workflow specification languages like CWL24 and WDL25. These define a declarative syntax for specifying workflows, which can be parsed and executed by arbitrary executors, e.g. Cromwell (https://cromwell.readthedocs.io), Toil26, and Tibanna27. Similar to the fourth niche, a downside is that imperative or functional programming is not or less integrated into the specification language, thereby limiting the expressive power. In contrast, a main advantage is that the same workflow definition can be executed on various specialized execution backends, thereby promising scalability to virtually any computing platform. Another important use case for system-independent languages is that they promote interoperability between other workflow definition languages. For example, Snakemake workflows can (within limits) be automatically exported to CWL, and Snakemake can make use of CWL tool definitions. An automatic translation of any CWL workflow definition into a Snakemake workflow is planned as well.\n\nToday, several of the above mentioned approaches support full in silico reproducibility of data analyses (e.g. Galaxy, Nextflow, Snakemake, WDL, CWL), by allowing the definition and scalable execution of each involved step, including deployment of the software stack needed for each step (e.g. via the Conda package manager, https://docs.conda.io, Docker, https://www.docker.com, or Singularity28 containers).\n\nReproducibility is important to generate trust in scientific results. However, we argue that a data analysis is only of lasting and sustained value for the authors and the scientific field if a hierarchy of additional interdependent properties is ensured (Figure 1).\n\nBy supporting the top layer, a workflow management system can promote the center layer, and thereby help to obtain true sustainability.\n\nFirst, to gain full in silico reproducibility, a data analysis has to be automated, scalable to various computational platforms and levels of parallelism, and portable in the sense that it is able to be automatically deployed with all required software in exactly the needed versions.\n\nSecond, while being able to reproduce results is a major achievement, transparency is equally important: the validity of results can only be fully assessed if the parameters, software, and custom code of each analysis step are fully accessible. On the level of the code, a data analysis therefore has to be readable and well-documented. On the level of the results it must be possible to trace parameters, code, and components of the software stack through all involved steps.\n\nFinally, valid results yielded from a reproducible data analysis have greater meaning to the scientific community if the analysis can be reused for other projects. In practice, this will almost never be a plain reuse, and instead requires adaptability to new circumstances, for example, being able to extend the analysis, replace or modify steps, and adjust parameter choices. Such adaptability can only be achieved if the data analysis can easily be executed in a different computational environment (e.g. at a different institute or cloud environment), thus it has to be scalable and portable again (see Figure 1). In addition, it is crucial that the analysis code is as readable as possible such that it can be easily modified.\n\nIn this work, we show how data analysis sustainability in terms of these aspects is supported by the open source workflow management system Snakemake (https://snakemake.github.io). Since its original publication in 2012, Snakemake has seen hundreds of releases and contributions (Figure 2c). It has gained wide adoption in the scientific community, culminating in, on average, more than five new citations per week, and over 700 citations in total (Figure 2a,b). This makes Snakemake one of the most widely used workflow management systems in science.\n\n(a) citations by year of the original Snakemake article (note that the year 2020 is still incomplete at the time of writing). (b) citations by scientific discipline of the citing article. Data source: https://badge.dimensions.ai/details/id/pub.1018944052, 2020/09/29. (c) cumulative number of git commits over time; Releases are marked as circles.\n\n\n2 Methods and results\n\nWe present how Snakemake enables the researcher to conduct data analyses that have all the properties leading to reproducibility, transparency and adaptability. This in turn allows the analysis to become a sustainable resource both for the researcher themselves and the scientific community. We structure the results by each of the properties leading to sustainable data analyses (Figure 1).\n\nWe will thereby introduce relevant features of both the workflow definition language as well as the execution environment. Several of them are shared with other tools, while others are (at the time of writing) exclusive to Snakemake. Finally, there are features that other workflow management systems provide while Snakemake does not (or not yet) offer them. We intentionally refrain from performing a full comparison with other tools, as we believe that such a view will never be unbiased (and quickly outdated), and should instead be provided by review articles or performed by the potential users based on their individual needs.\n\nThe central idea of Snakemake is that workflows are specified through decomposition into steps represented as rules (Figure 3). Each rule describes how to obtain a set of output files from a set of input files. This can happen via a shell command, a block of Python code, an external script (Python, R, or Julia), a Jupyter notebook (https://jupyter.org), or a so-called wrapper (see Sec. 2.2.1). Depending on the computing platform used and how Snakemake is configured, input and output files are either stored on disk, or in a remote storage (e.g. FTP, Amazon S3, Google Storage, Microsoft Azure Blob Storage, etc.). Through the use of wildcards, rules can be generic. For example, see the rule select_by_country in Figure 3a (line 20). It can be applied to generate any output file of the form results/by-country/{country}.csv, with {country} being a wildcard that can be replaced with any non-empty string. In shell commands, input and output files, as well as additional parameters, are directly accessible by enclosing the respective keywords in curly braces (in case of more than a single item in any of these, access can happen by name or index).\n\n(a) workflow definition; hypothesized knowledge requirement for line readability is color-coded on the left next to the line numbers. (b) directed acyclic graph (DAG) of jobs, representing the automatically derived execution plan from the example workflow; job node colors reflect rule colors in the workflow definition. (c) content of script plot-hist.py referred from rule plot_histogram. (d) knowledge requirements for readability by statement category (see subsection 3.2). The example workflow downloads data, plots histograms of city populations within a given list of countries, and converts these from SVG to PDF format. Note that this is solely meant as a short yet comprehensive demonstration of the Snakemake syntax.\n\nWhen using script integration instead of shell commands, Snakemake automatically inserts an object giving access to all properties of the job (e.g. snakemake.output [0], see Figure 3c). This avoids the presence and repetition of boiler plate code for parsing command line arguments. By replacing wildcards with concrete values, Snakemake turns any rule into a job which will be executed in order to generate the defined output files.\n\nDependencies between jobs are implicit, and inferred automatically in the following way. For each input file of a job, Snakemake determines a rule that can generate it— for example by replacing wildcards again (ambiguity can be resolved by prioritization or constraining wildcards)— yielding another job. Then, Snakemake goes on recursively for the latter, until all input files of all jobs are either generated by another job or already present in the used storage (e.g., on disk). Where necessary, it is possible to provide arbitrary Python code to infer input files based on wildcard values or even the contents of output files generated by upstream jobs.\n\nFrom this inference, Snakemake obtains a directed acyclic graph of jobs (DAG, see Figure 3b). The time needed for this is linear in the number of jobs involved in the workflow, and negligible compared to the usual runtimes of the workflow steps (see subsection 3.4).\n\nFigure 3a illustrates all major design patterns needed to define workflows with Snakemake: workflow configuration (line 1), aggregations (line 5–8), specific (line 33–43) and generic (line 45–53) transformations, target rules (line 3–8), log file definition, software stack definition, as well as shell command, script, and wrapper integration. subsection 3.1 presents additional patterns that are helpful in certain situations (e.g. conditional execution, iteration, exploration of large parameter spaces, benchmarking, scatter/gather).\n\n2.1.1 Automated unit test generation. When maintaining and developing a production workflow, it is important to test each contained step, ideally upon every change to the workflow code. In software development, such tests are called unit tests29. From a given source workflow with already computed results that have been checked for correctness, Snakemake can automatically generate a suite of unit tests, which can be executed via the Pytest framework (https://pytest.org). Each unit test consists of the execution of one rule, using input data taken from the source workflow. The generated results are by default compared byte-by-byte against the results given by in the source workflow. However, this behavior can be overwritten by the user. It is advisable to keep the input datasets of the source workflow small in order to ensure that unit tests finish quickly.\n\nThe workflow definition language of Snakemake is designed to allow maximum readability, which is crucial for transparency and adaptability. For natural-language readability, the occurrence of known words is important. For example, the Dale-Chall readability formula derives a score from the fraction of potentially unknown words (that do not occur in a list of common words) among all words in a text30. For workflow definition languages, one has to additionally consider whether punctuation and operator usage is intuitively understandable. When analyzing the above example workflow (Figure 3a) under these aspects, code statements fall into seven categories (subsection 3.2). In addition, for each statement, we can judge whether it\n\n1. needs domain knowledge (from the field analyzed in the given workflow),\n\n2. needs technical knowledge (e.g. about Unix-style shell commands or Python),\n\n3. needs Snakemake knowledge,\n\n4. is trivial (i.e., it should be understandable for everybody).\n\nIn Figure 3, we hypothesize the required knowledge for readability of each code line. Most statements are understandable with either general education, domain, or technical knowledge. In particular, only five lines need Snakemake-specific knowledge (Figure 3d). The rationale for each hypothesis can be found in subsection 3.2.\n\nWhile this example is obviously not as evolved as a realworld data analysis, the ratio of lines requiring Snakemake knowledge, and lines that are trivial or readable with domain or technical knowledge can be expected to stay roughly the same. Since Snakemake supports modularization of workflow definitions, it is moreover possible to hide more technical parts of the workflow definition (e.g. helper functions or variables), in order to not distract the reader from understanding the main steps of the data analysis.\n\nSince dependencies between jobs are implicitly encoded via matching filename patterns, we hypothesize that, in general, no specific technical knowledge is necessary to understand the connections between the rules (except for special cases like conditional or dynamically determined dependencies). The file-centric description of workflows makes it intuitive to to infer dependencies between steps; when the input of one rule reoccurs as the output of another, their link and order of execution is clear.\n\n2.2.1 Modularization. Specific data analysis steps can become quite complicated, for example, when plotting figures or working around idiosyncrasies of external tools. It helps readability to modularize these away such that the reader of a workflow only has to inspect them if interested in the specific step. Some of these steps can be quite specific and unique to the analysis. Others can be common to the scientific field and utilize widely used tools or libraries in a relatively standard way. For the latter, Snakemake provides the ability to deposit and use tool wrappers in/from a central repository. In contrast, the former can require custom code, often written in scripting languages like R or Python. Snakemake allows the user to modularize such steps either into scripts or to craft them interactively by integrating with Jupyter notebooks (https://jupyter.org).\n\nScript integration. Integrating a script works via a special script directive (see Figure 3a, line 42). The referred script does not need any boilerplate code, and can instead directly use all properties of the job (input files, output files, wildcard values, parameters, etc.), which are automatically inserted as a global snakemake object before the script is executed (see Figure 3c).\n\nJupyter notebook integration. Analogous to script integration, a notebook directive allows a rule to specify a path to a Jupyter notebook. Via the command line interface, it is possible to instruct Snakemake to open a Jupyter notebook server for editing a notebook in the context of a specific job derived from the rule that refers to the notebook. The notebook server can be accessed via a web browser in order to interactively program the notebook until the desired results (e.g. a certain plot or figure) are created as intended. Upon saving the notebook, Snakemake generalizes it such that other jobs from the same rule can subsequently re-use it automatically without the need for another interactive notebook session.\n\nTool wrappers. Reoccurring tools or libraries can be shared between workflows via Snakemake tool wrappers (see Figure 3a, line 52–53). A central public repository (https://snakemake-wrappers.readthedocs.io) allows the community to share wrappers with each other. Each wrapper consists of a Python or R script that either uses libraries of the respective scripting language or calls a shell command. Moreover, each wrapper provides a Conda environment defining the required software stack, including tool and library versions (see subsection 2.3). Often, shell command wrappers contain some additional code that works around various idiosyncrasies of the wrapped tool (e.g. dealing with temporary directories or converting job properties into command line arguments). A wrapper can be used by simply copying and adapting a provided example rule (e.g. by modifying input and output file paths). Upon execution, the wrapper code and the Conda environment are downloaded from the repository and automatically deployed to the running system. In addition to single wrappers, the wrapper repository also offers pre-defined, tested combinations of wrappers that constitute entire sub-workflows for common tasks (called meta-wrappers). This is particularly useful for combinations of steps that reoccur in many data analyses. All wrappers are automatically tested to run without errors prior to inclusion in the repository, and upon each committed change.\n\n2.2.2 Standardized code linting and formatting. The readability of programming code can be heavily influenced by adhering to common style and best practices31. Snakemake provides automatic code formatting (via the tool snakefmt) of workflows, together with any contained Python code. In addition, Snakemake has a built in code linter that detects code violating best practices (e.g. missing directives, indentation issues, missing environment variables, etc.) and provides suggestions on how to improve the code.\n\nBeing able to deploy a data analysis workflow to an unprepared system depends on: (a) the ability to install the workflow management system itself, and (b) the ability to obtain and use the required software stack for each analysis step. Snakemake itself is easily deployable via the Conda package manager (https://conda.io), as a Python package (https://pypi.io), or a Docker container (https://hub.docker.com/r/snakemake/snakemake). Instructions and further information can be found at https://snakemake.github.io.\n\nThe management of software stacks needed for individual rules is directly integrated into Snakemake itself, via two complementary mechanisms.\n\nConda integration For each rule, it is possible to define a software environment that will be automatically deployed via the Conda package manager (via a conda directive, see Figure 3a, line 15). Each environment is described by a lightweight YAML file used by conda to install constituent software. While efficiently sharing base libraries like Glib with the underlying operating system, software defined in the environment takes precedence over the same software in the operating system, and is isolated and independent from the same software in other Conda environments.\n\nContainer integration Instead of defining Conda environments, it is also possible to define a container for each rule (via a container directive, see Figure 3a, line 38). Upon execution, Snakemake will pull the requested container image and run a job inside that container using Singularity28. The advantage of using containers is that the execution environment can be controlled down to the system libraries, and becomes portable across operating systems, thereby further increasing reproducibility32. Containers already exist in centralized repositories for a wide range of scientific software applications, allowing easy integration info Snakemake workflows. The downside of using containers is that generating and modifying container images requires additional effort, as well as storage, since the image has to be uploaded to a container registry. The storage needed for containers can be minimized during workflow execution by reusing a common container image across multiple workflow steps, or by using step-specific containers comprised of small layers with step-specific customizations on top of a common shared base layer. A sweet spot between containers and Conda can be exploited by combining container and conda directives. In that case, Snakemake will generate the requested Conda environment inside of the container, providing the flexibility of Conda together with the additional reproducibility guarantees of containers.\n\nWhile processing a workflow, Snakemake tracks input files, output files, parameters, software, and code of each executed job. After completion, this information can be made available via self-contained, interactive, HTML based reports. Output files in the workflow can be annotated for automatic inclusion in the report. These features enable the interactive exploration of results alongside information about their provenance. Since results are included into the report, their presentation does not depend on availability of server backends, making Snakemake reports easily portable and archivable. In the future, Snakemake reports will be extended to additionally follow the RO-crate standard, which will make them machine-readable and allow an integration with web services like https://workflowhub.eu. An example report summarizing the data analysis conducted for this article can be found at https://doi.org/10.5281/zenodo.424414333.\n\nBeing able to scale a workflow to available computational resources is crucial for reproducing previous results as well as adapting a data analysis to novel research questions or datasets. Like many other state-of-the-art workflow management systems, Snakemake allows workflow execution to scale to various computational platforms, ranging from single workstations to large compute servers, any common cluster middleware, grid computing, and cloud computing (with native support for Kubernetes, the Google Cloud Life Sciences API, Amazon AWS, TES (https://www.ga4gh.org), and Microsoft Azure, the latter two in an upcoming release).\n\nSnakemake’s design ensures that scaling a workflow to a specific platform should only entail the modification of command line parameters. The workflow itself can remain untouched. Via configuration profiles, it is possible to persist and share the command line setup of Snakemake for any computing platform (https://github.com/snakemake-profiles/doc).\n\n2.5.1 Job scheduling. Because of their dependencies, not all jobs in a workflow can be executed at the same time. Instead, one can imagine partitioning the DAG of jobs into three sections: those that are already finished, those that have already been scheduled but are not finished yet, and those that have not yet been scheduled (Figure 4a). Let us call the jobs in the latter partition Jo, the set of open jobs. Within Jo, all jobs that have only incoming edges from the partition of finished jobs (or no incoming edge at all) can be scheduled next. We call this the set J of pending jobs. The scheduling problem a workflow manager like Snakemake has to solve is to select the subset E ⊆ J that leads to an efficient execution of the workflow, while not exceeding the given resources like hard drive space, I/O capacity and CPU cores. Snakemake solves the scheduling problem at the beginning of the workflow execution and whenever a job has finished and new jobs become pending.\n\n(a) Example workflow DAG. The greenish area depicts the jobs that are ready for scheduling (because all input files are present) at a given time during the workflow execution. We assume that the red job at the root generates a temporary file, which may be deleted once all blue jobs are finished. (b) Suboptimal scheduling solution: two green jobs are scheduled, such that only one blue job can be scheduled and the temporary file generated by the red job has to remain on disk until all blue jobs are finished in a subsequent scheduling step. (c) Optimal scheduling solution: the three blue jobs are scheduled, such that the temporary file generated by the red job can be deleted afterwards.\n\nEfficiency of execution is evaluated according to three criteria. First, execution should be as fast as possible. Second, high-priority jobs should be preferred (Snakemake allows prioritization of jobs via the workflow definition and the command line). Third, temporary output files should be quickly deleted (Snakemake allows output files to be marked as temporary, which leads to their automatic deletion once all consuming jobs have been finished). An example is shown in Figure 4.\n\nWe solve the scheduling problem via a mixed integer linear program (MILP) as follows. Let R be the set of resources used in the workflow (e.g., CPU cores and memory). By default, Snakemake only considers CPU cores which we indicate with c, i.e., R = {c}. Let F be the set of temporary files that are currently present. We first define constants for each pending job j ∈ J: Let pj ∈ ℕ be its priority, let ur,j ∈ ℕ be its usage of resource r ∈ R, and let zf,j ∈ {0, 1} indicate whether it needs temporary file f ∈ F as input (zf,j = 1) or not (zf,j = 0). Further, let Ur be the free capacity of resource r ∈ R (initially what is provided to Snakemake on the command line; later what is left, given resources already used in running jobs). Let Sf be the size of file f ∈ F, and let S:=∑f∈FSf be be total temporary file size (measured in some reasonable unit, such as MB).\n\nNext, we define indicator variables xj ∈ {0, 1}, for each job j ∈ J, indicating whether a job is selected for execution (1) or not (0). For each temporary file f ∈ F, we define a variable δf ∈ [0, 1] indicating the fraction of consuming jobs that will be scheduled among all open jobs. We also call this variable the lifetime fraction of temporary file f . In other words, δf = 1 means that all consuming jobs will be completed after this scheduling round has been processed, such that the lifetime of that file is over and it can be deleted. To indicate the latter, we further define a binary variable γf ∈ {0, 1}, with γf = 1 representing the case that f can indeed be deleted, in other words, γf = 1 ⇔ δf = 1.\n\nThen, the scheduling problem can be written as the MILP depicted in Table 1. The maximization optimizes four criteria, represented by four separate terms in (1). First, we strive to prefer jobs with high priority. Second, we aim to maximize the number of used cores, i.e. the extent of parallelization. Third, we aim to delete existing temporary files as soon as possible. Fourth, we try to reduce the lifetime of temporary files that cannot be deleted in this pass.\n\nWe consider these four criteria in lexicographical order. In other words, priority is most important, only upon ties do we consider parallelization. Given ties while optimizing parallelization, we consider the ability to delete temporary files. And only given ties when considering the latter, we take the lifetime of all temporary files that cannot be deleted immediately into account. Technically, this order is enforced by multiplying each criterion sum with a value that is at least as high as the maximum value that the equation right of it can acquire. Unless the user explicitly requests otherwise, all jobs have the same priority, meaning that in general the optimization problem maximizes the number of used cores while trying to remove as many temporary files as possible.\n\nThe constraints (2)–(4) ensure that the variables have the intended meaning and that the computed schedule does not violate resource constraints. Constraint (2) ensures that the available amount Ur of each resource r ∈ R is not exceeded by the selection. Constraint (3) (together with the fact that δf is being maximized) ensures that δf is ineed the lifetime fraction of temporary file f ∈ F. Note that the sum in the denominator extends over all open jobs, while the numerator only extends over pending jobs. Constraint (4) (together with the fact that γf is being maximized) ensures that γf = 0 if and only if δf < 1 and hence calculates whether temporary file f ∈ F can be deleted.\n\nAdditional considerations and alternatives, which may be implemented in subsequent releases of Snakemake, are discussed in subsection 3.3.\n\n2.5.2 Caching between workflows. While data analyses usually entail the handling of multiple datasets or samples that are specific to a particular project, they often also rely on retrieval and post-processing of common datasets. For example, in the life sciences, such datasets include reference genomes and corresponding annotations. Since such datasets potentially reoccur in many analyses conducted in a lab or at an institute, re-executing the analysis steps for retrieval and post-processing of common datasets as part of individual analyses would waste both disk space and computation time.\n\nHistorically, the solution in practice was to compile shared resources with post-processed datasets that could be referred to from the workflow definition. For example, in the life sciences, this has led to the Illumina iGenomes resource (https://support.illumina.com/sequencing/sequencing_software/igenome.html) and the GATK resource bundle (https://gatk.broadinstitute.org/hc/en-us/articles/360035890811-Resource-bundle).\n\nIn addition, in order to provide a more flexible way of selection and retrieval for such shared resources, so-called “reference management” systems have been published, like Go Get Data (https://gogetdata.github.io) and RefGenie (http://refgenie.databio.org). Here, the logic for retrieval and post-processing is curated in a set of recipes or scripts, and the resulting resources can be automatically retrieved via command line utilities. The downside of all these approaches is that the transparency of the data analysis is hampered since the steps taken to obtain the used resources are hidden and less accessible for the reader of the data analysis.\n\nSnakemake provides a new, generic approach to the problem which does not have this downside (see Figure 5). Leveraging workflow-inherent information, Snakemake can calculate a hash value for each job that unambiguously captures exactly how an output file is generated, prior to actually generating the file. This hash can be used to store and lookup output files in a central cache (e.g., a folder on the same machine or in a remote storage). For any output file in a workflow, if the corresponding rule is marked as eligible for caching, Snakemake can obtain the file from the cache if it has been created before in a different workflow or by a different user on the same system, thereby saving computation time, as well as disk space (on local machines, the file can be linked instead of copied).\n\nIf a job is eligible for caching, its code, parameters, raw input files, software environment and the hashes of its dependencies are used to calculate a SHA-256 hash value, under which the output files are stored in a central cache. Subsequent runs of the same job (with the same dependencies) in other workflows can skip the execution and directly take the output files from the cache.\n\nThe hash value is calculated in the following way. Let J be the set of jobs of a workflow. For any job j ∈ J, let cj denote its code (shell command, script, wrapper, or notebook), let Pj = {(ki, vi) \| i = 0,...,m} be its set of parameters (with key ki and JSON-encoded value vi), let Fj be its set of input files that are not created by any other job, and let sj be a string describing the associated software environment (either a container unique resource identifier, a Conda environment definition, or both). Then, assuming that job j ∈ J with dependencies Dj ⊂ J is the job of interest, we can calculate the hash value as\n\nwith h′ being the SHA-25634 hash function, ⊙ being the string concatenation, and ⊙ being the string concatenation of its operands in lexicographic order.\n\nThe hash function h(j) comprehensively describes everything that affects the content of the output files of job j, namely code, parameters, raw input files, the software environment and the input generated by jobs it depends on. For the latter, we recursively apply the hash function h again. In other words, for each dependency j′ ∈ Dj we include a hash value into the hash of job j, which is in fact the hashing principle behind blockchains used for cryptocurrency35. The hash is only descriptive if the workflow developer ensures that the cached result is generated in a deterministic way. For example, downloading from a URL that yields data which may change over time should be avoided.\n\n2.5.3 Graph partitioning. A data analysis workflow can contain diverse compute jobs, some of which may be long-running, and some which may complete quickly. When executing a Snakemake workflow in a cluster or cloud setting, by default, every job will be submitted separately to the underlying queuing system. For short-running jobs, this can result in a considerable overhead, as jobs wait in a queue, and may also incur additional delays or cost when accessing files from remote storage or network file systems. To minimize such overhead, Snakemake offers the ability to partition the DAG of jobs into subgraphs that will be submitted together, as a single cluster or cloud job.\n\nPartitioning happens by assigning rules to groups (see Figure 6). Upon execution, Snakemake determines connected subgraphs with the same assigned group for each job and submits such subgraphs together (as a so called group job) instead of submitting each job separately. For each group, it is in addition possible to define how many connected subgraphs shall be spanned when submitting (one by default). This way, it is possible to adjust the partition size to the needs of the available computational platform. The resource usage of a group job is determined by sorting involved jobs topologically, summing resource usage per level and taking the maximum over all levels.\n\nTwo rules of the example workflow (Figure 3a) are grouped together, (a) spanning one connected component, (b) spanning two connected components, and (c) spanning five connected components. Resulting submitted group jobs are represented as grey boxes.\n\n2.5.4 Streaming. Sometimes, intermediate results of a data analysis can be huge, but not important enough to store persistently on disk. Apart from the option to mark such files as temporary so that Snakemake will automatically delete them once no longer needed, it is also possible to instruct Snakemake to never store them on disk at all by directly streaming their content from the producing job to to the consuming job. This requires the producing and consuming jobs to run at the same time on the same computing node (then, the output of the producer can be written to a small in-memory buffer; on Unix, this is called a named pipe). Snakemake ensures this by submitting producer and consumer as a group job (see subsubsection 2.5.3).\n\n\n3 Further considerations\n\nFigure 7 shows advanced design patterns which are less common but useful in certain situations. For brevity, only rule properties that are necessary to understand each example are shown (e.g. omitting log directives and shell commands or script directives). Below, we explain each example in detail.\n\nFor brevity only rule properties that are necessary to understand each example are shown (e.g. omitting log directives and shell commands or script directives). (a) scatter/gather process, (b) streaming, (c) non-file parameters, (d) iteration, (e) sample sheet based configuration, (f) conditional execution, (g) benchmarking, (h) parameter space exploration. See subsection 3.1 for details.\n\nScatter/gather processes (Figure 7a). Snakemake’s ability to employ arbitrary Python code for defining a rule’s input and output files already enables any kind of scattering, gathering, and aggregations in workflows. Nevertheless, it can be more readable and scalable to use Snakemake’s explicit support for scatter/gather processes. A Snakemake workflow can have any number of such processes, each of which has a name (here someprocess). In this example, the rule scatter (line 4) splits some data into n items; the rule step2 (line 8) is applied to each item; the rule gather (line 14) aggregates over the outputs of step2 for each item. Thereby, n is defined via the scattergather directive (line 1) at the beginning, which sets n for each scatter/gather process in the workflow. In addition, n can be set via the command line via the flag --set-scatter. For example, here, we could set the number of scatter items to 16 by specifying --set-scatter someprocess=16. This enables the user to better scale the data analysis workflow to its computing platform, beyond the defaults provided by the workflow designer.\n\nStreaming (Figure 7b). Snakemake allows to stream output between jobs, instead of writing it to disk (see subsubsection 2.5.4). Here, the output of rule step1 (line 1) and step2 (line 7) is streamed into rule step3 (line 13).\n\nNon-file parameters (Figure 7c). Data analysis steps can need additional non-file input in the form of parameters, that are for example obtained from the workflow configuration (see subsection 2.1). Both input files and such non-file parameters can optionally be defined via a Python function, which is evaluated for each job, when wildcard values are known. In this example, we define a lambda expression (an anonymous function in Python), that retrieves a threshold depending on the value of the wildcard sample (w.sample, line 7). Wildcard values are passed as the first positional argument to such functions (here w, line 7).\n\nIteration (Figure 7d). Sometimes, a certain step in a data analysis workflow needs to be applied iteratively. Snakemake allows to model defining by setting the iteration count variable as a wildcard (here {i}, line 16). Then, an input function can be used to either request the output of the previous iteration (if i > 0, line 10) or the initial data (if i == 0, line 8). Finally, in the rule that requests the final iteration result, the wildcard {i} is set to the desired count (here 10, line 3).\n\nSample sheet based configuration (Figure 7e). Often, scientific experiments entail multiple samples, for which meta-information is known (e.g. gender, tissue etc. in biomedicine). Portable encapsulated projects (PEPs, https://pep.databio.org) are an approach to standardize such information and provide them in a shareable format. Snakemake workflows can be directly integrated with PEPs, thereby allowing to configure them via meta-information that is contained in the sample sheets defined by the PEP. Here, a pepfile (line 1) along with a validation schema (line 2) is defined, followed by an aggregation over all samples defined in the contained sample sheet.\n\nConditional execution (Figure 7f). By default, Snakemake determines the entire DAG of jobs upfront, before the first job is executed. However, sometimes the analysis path that shall be taken depends on some intermediate results. For example, this is the case when filtering samples based on quality control criteria. At the beginning of the data analysis, some quality control (QC) step is performed, which yields QC values for each sample. The actual analysis that shall happen afterwards might be only suitable for samples that pass the QC. Hence, one might have to filter out samples that do not pass the QC. Since the QC is an intermediate result of the same data analysis, it can be necessary to determine the part of the DAG that comes downstream of the QC only after QC has been finalized. Of course, one option is to separate QC and the actual analysis into two workflows, or defining a separate target rule for QC, such that it can be manually completed upfront, before the actual analysis is started. Alternatively, if QC shall happen automatically as part of the whole workflow, one can make use of Snakemake’s conditional execution capabilities. In the example, we define that the qc rule shall be a so-called checkpoint. Rules can depend on such checkpoints by obtaining their output from a global checkpoints object (line 2), that is accessed inside of a function, which is passed to the input directive of the rule (line 11). This function is re-evaluated after the checkpoint has been executed (and its output files are present), thereby allowing to inspect the content of the checkoint’s output files, and decide about the input files based on that. In this example, the checkpoint rule qc creates a TSV file, which the function loads, in order to extract only those samples for which the column \"some-value\" contains a value greater than 90 (line 6). Only for those samples, the file \"results/processed/{sample}.txt\" is requested, which is then generated by applying the rule process for each of these samples.\n\nBenchmarking (Figure 7g). Sometimes, a data analysis entails the benchmarking of certain tools in terms of runtime, CPU, and memory consumption. Snakemake directly supports such benchmarking by defining a benchmark directive in a rule (line 7). This directive takes a path to a TSV file. Upon execution of a job spawned from such a rule, Snakemake will constantly measure CPU and memory consumption, and store averaged results together with runtime information into the given TSV file. Benchmark files can be input to other rules, for example in order to generate plots or summary statistics.\n\nParameter space exploration (Figure 7h). In Python (and therefore also with Snakemake), large parameter spaces can be represented very well via Pandas36,37 data frames. When such a parameter space shall be explored by the application of a set of rules to each instance of the space (i.e., each row of the data frame), the idiomatic approach in Snakemake is to encode each data frame column as a wildcard and request all occuring combinations of values (i.e., the data frame rows), by some consuming rule. However, with large parameter spaces that have a lot of columns, the wildcard expressions could become cumbersome to write down explicitly in the Snakefile. Therefore, Snakemake provides a helper called Paramspace, which can wrap a Pandas data frame (this functionality was inspired by the JUDI workflow management system https://pyjudi.readthedocs.io). The helper allows to retrieve a wildcard pattern (via the property wildcard_pattern) that encodes each column of the data frame in the form name~{name} (i.e., column name followed by the wildcard/wildcard value). The wildcard pattern can be formatted into input or output file names of rules (line 15). The method instance of the Paramspace object, automatically returns the corresponding data frame row (as a Python dict) for given wildcard values (here, that method is automatically evaluated by Snakemake for each instance of the rule simulate, line 17). Finally, aggregation over a parameter space becomes possible via the property instance_patterns, which retrieves a concrete pattern of above form for each data frame row. Using the expand helper, these patterns can be formatted into a file path (line 8–11), thereby modelling an aggregation over the entire parameter space. Naturally, filtering rows or columns on the paramspace via the usual Pandas methods allows to generate sub-spaces.\n\nStatements in Snakemake workflow definitions fall into seven categories:\n\n1. a natural language word, followed by a colon (e.g. input: and output:),\n\n2. the word “rule”, followed by a name and a colon (e.g. ruleconvert_to_pdf:),\n\n3. a quoted filename pattern (e.g. \"{prefix}.pdf\"),\n\n4. a quoted shell command,\n\n5. a quoted wrapper identifier,\n\n6. a quoted container URL\n\n7. a Python statement.\n\nBelow, we list the rationale of our assessment for each category in Figure 3:\n\n1. The natural language word is either trivially understandable (e.g. input: and output:) or understandable with technical knowledge (container: or conda:). The colon straightforwardly shows that the content follows next. Only for the wrapper directive (wrapper:) one needs to have the Snakemake specific knowledge that it is possible to refer to publicly available tool wrappers.\n\n2. The word “rule” is trivially understandable, and when carefully choosing rule names, at most domain knowledge is needed for understanding such statements.\n\n3. Filename patterns can mostly be understood with domain knowledge, since the file extensions should tell the expert what kind of content will be used or created. We hypothesize that wildcard definitions (e.g. {country}) are straightforwardly understandable as a placeholder.\n\n4. Shell commands will usually need domain and technical knowledge for understanding.\n\n5. Wrapper identifiers can be understood with Snakemake knowledge only, since one needs to know about the central tool wrapper repository of Snakemake. Nevertheless, with only domain knowledge one can at least conclude that the mentioned tool (last part of the wrapper ID) will be used in the wrapper.\n\n6. A container URL will usually be understandable with technical knowledge.\n\n7. Python statements will either need technical knowledge or Snakemake knowledge (when using the Snakemake API, as it happens here with the expand command, which allows to aggregate over a combination of wildcard values).\n\nWhile the first releases of Snakemake used a greedy scheduler, the current implementation aims at using more efficient schedules by solving a mixed integer linear program (MILP) whenever there are free resources. The current implementation already works well; still, future releases may consider additional objectives:\n\nThe current formulation leads to fast removal of existing temporary files. In addition, one may control creation of temporary files in the first place, such that only limited space is occupied by temporary files at any time point during workflow execution.\n\nIt may also be beneficial to initially identify bottleneck jobs in the graph and prioritize them automatically instead of relying on the workflow author to prioritize them.\n\nBecause we consider different objectives hierarchically and use large constants in the objective function, currently a high solver precision is needed. If more objectives are considered in the future, an alternative hierarchical formulation may be used: First find the optimal objective value for the first (or the first two) objectives; then solve another MILP that maximizes less important objectives and ensures via constraints that the optimality of the most important objective(s) is not violated, or stays within, say, 5% of the optimal value.\n\nWe also need to mention a technical detail about the interaction between the scheduler and streams (subsection 3.1). Some jobs that take part in handling a data stream may effectively use zero cores (because they mostly wait for data and then only read or write data), i.e. they have uc,j = 0 in the MILP notation, which means that they do not contribute to the objective function. We thus replace the MILP objective term that maximizes paralellization (∑j∈Juc,j⋅xj) by the modified term ∑j∈Jmax⁡{uc,j,1}⋅xj to ensure that the weight of any xj within the sum is at least 1.\n\nWhen executing a data analysis workflow, running time and resource usage is dominated by the executed jobs and the performance of the libraries and tools used in these. Nevertheless, Snakemake has to process dependencies between jobs, which can incur some startup time until the actual workflow is executed. In order to provide an estimate on the amount of time and memory needed for this computation, we took the example workflow from Figure 3 in the main manuscript and artificially inflated it by replicating the countries in the input dataset. By this, we generated workflows of 10 to 90,000 jobs. Then, we benchmarked runtime and memory usage of Snakemake for computing the entire graph of jobs on these on a single core of an Intel Core i5 CPU with 1.6 GHz, 8 GB RAM and a Lenovo PCIe SSD (LENSE20512GMSP34MEAT2TA) (Figure 8). It can be seen that both runtime and memory increase linearly, starting from 0.2 seconds with 2.88 MB for 11 jobs and reaching 60 seconds with 1.1 GB for 90,000 jobs.\n\nThe Snakemake workflow generating the results along with a self-contained Snakemake report that connects results and provenance information is available at https://doi.org/10.5281/zenodo.4244143.\n\nFor future releases of Snakemake, we plan to further improve performance, for example by making use of PyPy (https://www.pypy.org), and by caching dependency resolution results between subsequent invocations of Snakemake.\n\n\n4 Conclusion\n\nWhile having been almost the holy grail of data analysis workflow management in recent years and being certainly of high importance, reproducibility alone is not enough to sustain the hours of work that scientists invest in crafting data analyses. Here, we outlined how the interplay of automation, scalability, portability, readability, traceability, and documentation can help to reach beyond reproducibility, making data analyses adaptable and transparent. Adaptable data analyses can not only be repeated on the same data, but also be modified and extended for new questions or scenarios, thereby greatly increasing their value for both the scientific community and the original authors. While reproducibility is a necessary property for checking the validity of scientific results, it is not sufficient. Being able to reproduce exactly the same figure on a different machine tells us that the analysis is robust and valid from a technical perspective. However, it does not tell anything about the methodological validity (correctness of statistical assumptions, avoidance of overfitting, etc.). The latter can only be secured by having a transparent yet accessible view on the analysis code.\n\nBy analyzing its readability and presenting its modularization, portability, reporting, scheduling, caching, partitioning, and streaming abilities, we have shown how Snakemake supports all these aspects, thereby providing a comprehensive framework for sustainable data analysis, and enabling an ergonomic, unified, combined representation of any kind of analysis step, from raw data processing, to quality control and fine-grained, interactive exploration and plotting of final results.\n\n\nSoftware availability\n\nSnakemake is available as MIT licensed open source software (homepage: https://snakemake.github.io, repository: https://github.com/snakemake/snakemake) and can be installed via Bioconda38.\n\n\nData availability\n\nThe Snakemake workflow generating the results presented in this work, along with the corresponding Snakemake report connecting results and provenance information is available at https://doi.org/10.5281/zenodo.424414333.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Author information\n\nFelix Mölder has designed and implemented the job scheduling mechanism (subsubsection 2.5.1; supervised by Johannes Köster and Sven Rahmann) and edited the manuscript. Kim Philip Jablonski has designed and implemented Jupyter notebook integration (subsubsection 2.2.1) and edited the manuscript. Michael Hall and Brice Letcher have designed and implemented automated code formatting (subsubsection 2.2.2) and Brice Letcher has edited the manuscript. Vanessa Sochat has designed and implemented the Google Cloud Life Sciences API execution backend, as well as various improvements to Google storage support (subsection 2.5) and edited the manuscript. Soohyun Lee has designed and implemented the AWS execution backend via integration with Tibanna (subsection 2.5). Sven O. Twardziok and Alexander Kanitz have designed and implemented the TES execution backend (subsection 2.5). Andreas Wilm has designed and implemented the Microsoft Azure execution backend (subsection 2.5) and edited the manuscript. Manuel Holtgrewe has designed and implemented bench-marking support (subsection 3.1). Jan Forster has designed and implemented meta-wrapper support (subsubsection 2.2.1). Christopher Tomkins-Tinch has designed and implemented remote storage support (subsection 2.1) and edited the manuscript. Sven Rahmann has edited the manuscript. Sven Nahnsen has provided the initial idea of using blockchain hashing to fingerprint output files a priori (subsubsection 2.5.2). Johannes Köster has written the manuscript and implemented all other features that occur in the text but are not explicitly mentioned in above listing. All authors have read and approved the manuscript.\n\n\nAcknowledgements\n\nWe are most grateful for the thousands of Snakemake users, their enhancement proposals, bug reports, and efforts to perform sustainable data analyses. We deeply thank all contributors to the Snakemake, Snakemake-Profile, Snakemake-Workflows, and Snakemake-Wrappers codebases.\n\n\nReferences\n\nBaker M: 1,500 scientists lift the lid on reproducibility. Nature. 2016; 533(7604): 452–4. 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Publisher Full Text\n\nThe pandas development team: pandas-dev/pandas: Pandas. Version latest. 2020. Publisher Full Text\n\nGrüning B, Dale R, Sjödin A, et al.: Bioconda: sustainable and comprehensive software distribution for the life sciences. Nat Methods. 2018; 15(7): 475–476. PubMed Abstract \| Publisher Full Text" }	[ { "id": "77869", "date": "15 Feb 2021", "name": "Michael Reich", "expertise": [ "Reviewer Expertise Bioinformatics", "reproducible research", "workflow systems", "open science" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors describe Snakemake, a text-based pipeline execution system that builds on the principles of the Unix 'make' utility to include optimized job scheduling, extensive specification of compute resources, advanced workflow features, integration with package managers and container technologies, and result caching. The authors identify where Snakemake exists within the large ecosystem of pipeline management systems and describe the motivating principles of Snakemake in terms of a hierarchy of sustainable data analysis that includes reproducibility, accessibility, transparency, and other objectives. They then walk through several examples illustrating the scope and use of the system. This paper updates and extends the original Snakemake publication of Köster et al., Bioinformatics 20121. The value of Snakemake to the field of bioinformatics is well-established, and the paper provides usage statistics and citations to reinforce this point.\nThe paper thoroughly describes the features of Snakemake and the necessary background to understand their use. While it is possible to understand a Snakemake workflow at a high level with little programming knowledge, experience with Unix, Python, and an understanding of how 'make' works are necessary to author Snakemake workflows. The authors balance the conceptual overview with well-chosen usage examples that are simple enough to understand and make clear how the example can generalize to other cases. They also describe in detail the job scheduling algorithm that snakemake uses. The paper would benefit from a brief discussion of how snakemake interacts with load-balancing software (such as SLURM, Torque, LSF, etc.), because of the focus on the details of executing jobs. This information is in the documentation on the Snakemake web site, but a conceptual overview in the paper would help the reader to understand this relationship.\nInformation necessary to install and use snakemake is available on the snakemake web site as referenced in the paper. The instructions and tutorial are comprehensive and understandable, and the progressive exercises give a good feel for the snakemake paradigm. Given the ease and popularity of container technology, it would be useful to include how to run snakemake from a Docker container in the tutorial. I was able to do this easily from the official snakemake container on Dockerhub and think it would be a helpful alternative in the documentation to installing Miniconda.\nIn general, this paper provides a comprehensive introduction and overview of Snakemake and serves as an effective entry point to this popular system.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "6549", "date": "19 Apr 2021", "name": "Johannes Köster", "role": "Author Response", "response": "Thanks a lot for the assessment of our manuscript. Your comments have led to several important improvements. Answers to individual comments can be found below. Comment 1 The paper would benefit from a brief discussion of how snakemake interacts with load-balancing software (such as SLURM, Torque, LSF, etc.), because of the focus on the details of executing jobs. This information is in the documentation on the Snakemake web site, but a conceptual overview in the paper would help the reader to understand this relationship. Response 1 Indeed, the paper failed to explicitly mention how this is handled. We have now added an explaining paragraph to section 2.5.1. In brief: resource requirements are passed to the middleware, but the scheduling problem still has to be solved in order to prioritze jobs and minimize the lifetime of temporary files. It becomes a bit less constrained though. Comment 2 Information necessary to install and use snakemake is available on the snakemake web site as referenced in the paper. The instructions and tutorial are comprehensive and understandable, and the progressive exercises give a good feel for the snakemake paradigm. Given the ease and popularity of container technology, it would be useful to include how to run snakemake from a Docker container in the tutorial. I was able to do this easily from the official snakemake container on Dockerhub and think it would be a helpful alternative in the documentation to installing Miniconda. Response 2 Offering further ways of performing the tutorial is indeed a good idea. We have considered providing instructions for performing the tutorial from a container, but felt that this would complicate editing and interaction too much. However, we now instead provide a Gitpod setup for the tutorial, which allows to directly run it, without any setup needed, from inside a browser window which offers both an editor and a terminal based on the Eclipse Theia IDE (see here)." } ] }, { "id": "79773", "date": "11 Mar 2021", "name": "Caroline C. Friedel", "expertise": [ "Reviewer Expertise Bioinformatics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present an updated version of their workflow management system snakemake. Here, the authors focus on particular aspects important for sustainable data analysis, in particular automation, readability, portability, documentation and scalability. This is followed by a section on \"further consideration\", which presents specific workflow patterns, more details on their readability analysis, some more details on scheduling as well as a performance analysis. Apart from the performance analysis at the end, these \"further considerations\" aspects would be more appropriate earlier in the manuscript. In particular, the scheduling considerations (3.3) would be more appropriate in the section on scalability (2.5), where scheduling is extensively described. The readability considerations (3.2) would be more appropriate in the readability section earlier (2.2), in particular as the latter refers extensively to these considerations. The current structure ironically reduces readability of the article.\nThe advanced workflow design patterns are more difficult to place, but would be more appropriate somewhere before readability as they are not quite as easy to read as the simple example at the beginning and their readability should be discussed. While the authors state that they are \"less common but useful in certain situations\", I would argue that some of them, in particular \"Non-file parameters\" (Fig. 7c) should be commonly used. Most bioinformatics tools one would want to use in a workflow, have multiple non-file parameters where one would not necessarily use the default values (if there even are defaults).\nApart from these issues, the authors present their case well on what they consider to be important for sustainable data analysis and show that snakemake is both well maintained, and commonly used, not only in the bioinformatics community. The section on job scheduling is very extensive, but I am also missing some details on how snakemake interacts with cluster scheduling software like SLURM etc. The question remains whether that complex mixed integer linear program for scheduling is still relevant or necessary if jobs will be submitted to a cluster with an own load-balancing software anyway. Another point that should be addressed is how or if different computing environments could be combined, e.g. if one has both a high-memory machine available for memory-intensive jobs and a separate computing cluster. Would one have to either run all jobs in the same environment or separate the workflow into two workflows that are run separately?\nThe main issue I have with the manuscript is that the authors overstate the readability of snakemake workflows. Readability is extensively discussed on a very simply workflow even with a sort of quantification of the readability of the workflow. However, this workflow would also be pretty easy to read as a simple bash script, so I do not think this is an appropriate example to show how readable snakemake workflows are. Furthermore, several of the lines they consider \"trivial\", in my opinion, still require some understanding of snakemake, make or programming. I have previously worked with snakemake, though not recently, and in my experience the rule structure is not easy to mentally parse if one is not familiar with it. Moreover, even if every single line were trivial the whole workflow could still be difficult to understand due to the dependencies which are implicitly created through use of common in- or output files. This can very easily lead to a very complex structure, in particular since the order in which rules are given does not have to be in order of their dependencies. Their argument is also somewhat contradicted by the advanced workflow design patterns presented later, which are not that easy to read even with the explanation.\nI think the article would benefit if instead of trying to quantify the readability of the simple workflow, the authors would focus more on the approaches they included in snakemake for improving readability of workflows, i.e. modularization and standardized code linting and formatting. In particular, I would be interested in hearing more details on the snakefmt tool and the recommended best practices.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "6551", "date": "19 Apr 2021", "name": "Johannes Köster", "role": "Author Response", "response": "Thanks a lot for the comprehensive assessment of the manuscript. This was very helpful and we are confident that your suggestions have significantly improved the article. Answers to individual comments can be found below. Comment 1 Here, the authors focus on particular aspects important for sustainable data analysis, in particular automation, readability, portability, documentation and scalability. This is followed by a section on \"further consideration\", which presents specific workflow patterns, more details on their readability analysis, some more details on scheduling as well as a performance analysis. Apart from the performance analysis at the end, these \"further considerations\" aspects would be more appropriate earlier in the manuscript. In particular, the scheduling considerations (3.3) would be more appropriate in the section on scalability (2.5), where scheduling is extensively described. The readability considerations (3.2) would be more appropriate in the readability section earlier (2.2), in particular as the latter refers extensively to these considerations. The current structure ironically reduces readability of the article. Response 1 Thanks a lot for this suggestion. We had indeed moved around these sections several times before submission. In the end, we thought the current structure is best at addressing a potentially diverse readership (technical and non-technical, seeking for a quick overview or for in-depth details, experienced users and beginners) with section 2 providing a comprehensive overview and section 3 showing additional details for particularly interested readers. We have added an explanation of the concept to the end of the introduction, and hope that this clarifies the intention and hopefully diminishes the negative effects of the split. Comment 2 The advanced workflow design patterns are more difficult to place, but would be more appropriate somewhere before readability as they are not quite as easy to read as the simple example at the beginning and their readability should be discussed. While the authors state that they are \"less common but useful in certain situations\", I would argue that some of them, in particular \"Non-file parameters\" (Fig. 7c) should be commonly used. Most bioinformatics tools one would want to use in a workflow, have multiple non-file parameters where one would not necessarily use the default values (if there even are defaults). Response 2 Thanks a lot for these important thoughts. Indeed, we agree that our initial example was lacking parameter definitions via the params directive, which are indeed quite ubiquitous in practice. We have extended our example accordingly. Comment 3 Apart from these issues, the authors present their case well on what they consider to be important for sustainable data analysis and show that snakemake is both well maintained, and commonly used, not only in the bioinformatics community. The section on job scheduling is very extensive, but I am also missing some details on how snakemake interacts with cluster scheduling software like SLURM etc. The question remains whether that complex mixed integer linear program for scheduling is still relevant or necessary if jobs will be submitted to a cluster with an own load-balancing software anyway. Response 3 Indeed, the scheduling problem description was lacking an explanation about what happens in a cluster/cloud setting. We have extended the text accordingly. In brief: resource requirements are passed to the cluster/cloud middleware, but the scheduling problem still has to be solved in order to prioritze jobs and minimize the lifetime of temporary files. It becomes a bit less constrained though. Comment 4 Another point that should be addressed is how or if different computing environments could be combined, e.g. if one has both a high-memory machine available for memory-intensive jobs and a separate computing cluster. Would one have to either run all jobs in the same environment or separate the workflow into two workflows that are run separately? Response 4 This is indeed a very good point. For addressing execution on multiple machines, Snakemake entirely relies on cluster or cloud middleware. By specifying resource requirements per rule (or dynamically per job), which are passed to the middleware, it is of course possible to run different jobs on different types of machines. What is currently not possible is to combine different execution backends like two different cluster systems or a cluster and a local high memory machine. We have updated the text accordingly. Comment 5 The main issue I have with the manuscript is that the authors overstate the readability of snakemake workflows. Readability is extensively discussed on a very simply workflow even with a sort of quantification of the readability of the workflow. However, this workflow would also be pretty easy to read as a simple bash script, so I do not think this is an appropriate example to show how readable snakemake workflows are. Furthermore, several of the lines they consider \"trivial\", in my opinion, still require some understanding of snakemake, make or programming. I have previously worked with snakemake, though not recently, and in my experience the rule structure is not easy to mentally parse if one is not familiar with it. Moreover, even if every single line were trivial the whole workflow could still be difficult to understand due to the dependencies which are implicitly created through use of common in- or output files. This can very easily lead to a very complex structure, in particular since the order in which rules are given does not have to be in order of their dependencies. Their argument is also somewhat contradicted by the advanced workflow design patterns presented later, which are not that easy to read even with the explanation. I think the article would benefit if instead of trying to quantify the readability of the simple workflow, the authors would focus more on the approaches they included in snakemake for improving readability of workflows, i.e. modularization and standardized code linting and formatting. In particular, I would be interested in hearing more details on the snakefmt tool and the recommended best practices. Response 5 This is indeed a valid point, thanks a lot for bringing it up. We have rewritten the readability section to better reflect that the presented example shows an ideal, quite simple situation, that might be impossible to reach for parts of workflows in practice (but nevertheless should be aimed for). We have added advice on how to use modularization to help the reader of a workflow in such cases. We still think that the example is appropriate (in combination with the mentioned changes in the text). While it is indeed simple, a bash script that would contain all the work that Snakemake is doing behind the scenes (checking file consistency, scheduling, various execution backends, parallelization, software stack deployment, etc.) would be much longer and less readable. We have also checked the triviality claims in all lines again (the numbers did change slightly). Of course, we'd be grateful to discuss or directly modify specific examples where you might still disagree with our judgement after the fixes. We agree that the dependency structure can be sometimes complicated and we have therefore added some sentences that clarify this. Here, it is important to mention Snakemake's ability to visualize dependencies and to automatically generate interactive reports. Since the latter are of particular value for peeking into the codebase without needing to understand the entire workflow, we have further extended the corresponding section (2.4). We are thankful for the suggestion to elaborate on other measures for improving readability and have therefore extended our section on the linter and formatter (Section 2.2.2). Further, we have added some additional sentences about modularization and how to use it best for ensuring readability (Section 2.2.1)." } ] } ]	1	https://f1000research.com/articles/10-33
https://f1000research.com/articles/10-103/v1	11 Feb 21	{ "type": "Method Article", "title": "ASaiM-MT: a validated and optimized ASaiM workflow for metatranscriptomics analysis within Galaxy framework", "authors": [ "Subina Mehta", "Marie Crane", "Emma Leith", "Bérénice Batut", "Saskia Hiltemann", "Magnus Ø Arntzen", "Benoit J. Kunath", "Francesco Delogu", "Ray Sajulga", "Praveen Kumar", "James E. Johnson", "Timothy J. Griffin", "Pratik D. Jagtap", "Marie Crane", "Emma Leith", "Bérénice Batut", "Saskia Hiltemann", "Magnus Ø Arntzen", "Benoit J. Kunath", "Francesco Delogu", "Ray Sajulga", "Praveen Kumar", "James E. Johnson", "Timothy J. Griffin" ], "abstract": "The Human Microbiome Project (HMP) aided in understanding the role of microbial communities and the influence of collective genetic material (the ‘microbiome’) in human health and disease. With the evolution of new sequencing technologies, researchers can now investigate the microbiome and map its influence on human health. Advances in bioinformatics methods for next-generation sequencing (NGS) data analysis have helped researchers to gain an in-depth knowledge about the taxonomic and genetic composition of microbial communities. Metagenomic-based methods have been the most commonly used approaches for microbiome analysis; however, it primarily extracts information about taxonomic composition and genetic potential of the microbiome under study, lacking quantification of the gene products (RNA and proteins). Conversely, metatranscriptomics, the study of a microbial community’s RNA expression, can reveal the dynamic gene expression of individual microbial populations and the community as a whole, ultimately providing information about the active pathways in the microbiome. In order to address the analysis of NGS data, the ASaiM analysis framework was previously developed and made available via the Galaxy platform. Although developed for both metagenomics and metatranscriptomics, the original publication demonstrated the use of ASaiM only for metagenomics, while thorough testing for metatranscriptomics data was lacking. In the current study, we have focused on validating and optimizing the tools within ASaiM for metatranscriptomics data. As a result, we deliver a robust workflow that will enable researchers to understand dynamic functional response of the microbiome in a wide variety of metatranscriptomics studies. This improved and optimized ASaiM-metatranscriptomics (ASaiM-MT) workflow is publicly available via the ASaiM framework, documented and supported with training material so that users can interrogate and characterize metatranscriptomic data, as part of larger meta-omic studies of microbiomes.", "keywords": [ "Galaxy", "metatranscriptomics", "microbiome", "functional analysis" ], "content": "Introduction\n\nUnderstanding the role of microbiome in patho-physiological conditions such as inflammatory diseases, obesity, and cancer has opened up various avenues of research1.Experimental design and biological interpretation of microbiome data has become an area of intense focus as the contributions to human health and disease are becoming clearer2,3. The ‘meta-omics’ approaches, such as metagenomics, metatranscriptomics and metaproteomics have been developed to study microbiomes without culturing and target the major macromolecules that constitute the community, namely DNA, RNA and proteins. While metagenomics (16S rRNA or whole genome sequencing) focuses on the taxonomy profile and functional potential4, metatranscriptomics, metaproteomics and meta-metabolomics5 uncover the functional response of the microbiome to stimuli on the short and long time-scale, respectively6,7.\n\nMetatranscriptomics has been used to analyze microbial gene expression profiles from a variety of complex sample types, e.g. human microbiome, aquatic or terrestrial environments, plant-microbe interactions8. Despite these applications, challenges still exist in the analysis of the complex metatranscriptomics data. Metatranscriptomics data is usually generated using high-throughput sequencing of short RNA-Seq reads using Illumina sequencing technology (PMID: 30298254). Many software tools and workflows are available for metatranscriptomics analysis. These include tools for RNA-Seq Data Preprocessing: Quality Control (FastQC), Ribosomal RNA removal (SortMeRNA, barrnap), host RNA removal (BMTagger), De Novo Assembly (Trinity, MetaVelvet, Oases, IDBA-MT, TAG), Transcript Taxonomy (Kraken, GOTTCHA, MetaPhlAn2), Functional Annotation (HUMAnN2), Annotation of assembled contigs are subjected to gene finding programs such as FragGeneScan followed by functional assignment using DIAMOND searches against KEGG, NCBI RefSeq, UniProt. Differential Expression analysis is performed by tools such as EdgeR, DeSeq2 and limma. “Reads-Based” analysis is performed by tools such as MetaTrans, COMAN, FMAP, SAMSA2, ASaiM and Assembly Based analysis: SqueezeMeta, IMP, MOSCA. Some of these open source tools9 have been incorporated within the Galaxy bioinformatics workbench10 to make it more accessible to users on a single platform.\n\nASaiM framework was previously developed by Batut et al. to perform metagenomics and metatranscriptomics data analysis11. The major goal of ASaiM was to develop an accessible, resharable, and user-friendly framework for microbiome researchers, implemented within the Galaxy platform12. The framework integrates a comprehensive set of microbiota related tools, predefined and tested workflows as well as supporting training material and documentation. It is available for users as a Docker image but also as a web server (https://metagenomics.usegalaxy.eu/). This implementation also enables flexibility, so that the workflow can be customized for datasets of diverse origin as new software tools or methods emerge. To address the need for optimizing ASaiM for metatranscriptomics data, we added the ASaiM-metatranscriptomics (ASaiM-MT) (Figure 1), a metatranscriptomics workflow, and rigorously tested it to ensure reliable analysis of metatranscriptomics data. Our testing and validation focused on using contemporary tools in their most current version (Table 1), capable of handling large datasets, and ensuring that the outputs from each of the tools were compatible in order to build an integrated and automated workflow. The workflow also has potential for integration with other meta-omic tools and workflows in Galaxy, such as those designed for metaproteomics13, to enable multi-omic data analysis.\n\n(i) Preprocessing: Process raw metatranscriptomics data to perform further analysis. (ii) Taxonomy Quantitation: Assignment of taxonomy along with abundance values and visualization. (iii) Functional Quantitation: metabolic assignment of identified functions and gene and pathway abundance annotation. (iv) Taxonomy-Function Quantitation: combine taxonomy and functional quantitation values into relative abundance values at different levels such as e.g., the abundance of a pathway between phyla.\n\nThe ASaiM-MT workflow is available via the ASaiM framework, specially at https://metagenomics.usegalaxy.eu/), for users to test their metatranscriptomics data. It is supported by a step-by-step tutorial (link), available on the Galaxy Training Network (GTN) (citation), which provides explanation for the different steps and the opportunity for online, hands-on training in using the workflow, with a trimmed dataset.\n\n\nMethods\n\nOur optimized ASaiM-MT workflow (available via https://metagenomics.usegalaxy.eu/) accepts Illumina paired end FASTQ sequence files (Forward read and Reverse read). As an alternative, a single-end FASTQ sequence can also be used as an input, with minor modifications in the downstream processing tool (such as changing the sequence type in CutAdapt and Filter with SortmeRNA as single end reads and also removing the FASTQ interlacer tool).\n\nThe ASaiM-MT workflow contains all the processing steps and the parameters required for the metatranscriptomics analysis from RNA-Seq data collected under a single biological condition. This workflow is also compatible with the single-end sequencing reads although parameters have to be changed to accommodate this input. This workflow is a multi-step analysis with preprocessing/ data cleaning, taxonomy analysis and functional analysis. The starting data input for the workflow are the FASTQ files - forward and reverse reads (obtained from the Illumina sequencer). We describe below the tools and their functions in the workflow. For comparative analysis of multiple biological conditions, the users have an option to use the MT2MQ tool to generate inputs for statistical analysis (see discussion).\n\n1) Preprocessing\n\nOccasionally, sequencing can introduce incorrect identification of nucleotides and these errors can lead to misinterpretation of the data, thus bringing in the need to preprocess (Figure 2(a)) the data before analysis. The first step in our analysis is to perform quality control to remove such sequencing errors. For this, we use FastQC to assess the quality of each sample and MultiQC to combine each result into a single report. To improve the quality of the data, CutAdapt was used to trim low-certainty bases from reads, filter out reads of poor quality or short length, unwanted sequences, including adapters, primers, and poly-A tails. The ASaiM-metagenomics shotgun workflow uses Trim Galore! for trimming of adapters. Trim Galore! works as a wrapper that includes CutAdapt and FastQC. However, for the ASaiM-MT workflow we chose CutAdapt14 for adapter trimming because it is more error tolerant, processes fast and modifies and filters reads according to user’s preference compared to TrimGalore!. Next, SortMeRNA15 was used to remove any rRNA sequences, which are often used for easy taxonomic characterization of microbiomes but do not provide functional information. We eliminated the step of de-replication of reads (V-Search) in the ASaiM-MT workflow, in order to retain the multiple copies of sequences for metatranscriptomics quantitation. The final step in cleaning and processing the data is to interlace the forward and reverse reads since the following steps require a single file per sample. For performing this action, the original ASaiM Shotgun workflow used the FASTQ-joiner to join the reads. However, in the ASaiM-MT version, we use the FASTQ interlacer. FASTQ interlacer joins the forward (/1) and the reverse reads (/2) using the sequence identifiers; sequences without designation will be named as single reads. The reason ASaiM-MT uses FASTQ-interlacer rather than FASTQ-joiner is because the joiner tool combines the forward and reverse read sequence together while the interlacer puts the forward and reverse read sequences in the same file while retaining the entity of each read along with an additional file with unpaired sequences. We perform the interlacing on the data both before and after the SortMeRNA step since the following steps require both data with and data without rRNA.\n\na) Preprocessing workflow: Workflow representation of the tools involved in quality check, data trimming and RNA filtering. b) Taxonomic profile workflow: workflow representation of taxonomy assignment tool (MetaPhlAn2) and post processing of the data using the Format MetaPhlAn2 tool. The workflow includes visualization of the data using interactive Krona and GraPhlAn plots. c) Functional information workflow: representation of tools involved in functional annotation (HUMAnN2), normalization of the data d) Combine Functional-Taxonomy abundance workflow: workflow representing tools that combines (Combine MetaPhlAn2 and HUMAnN2 outputs) and groups (Group abundances into GO slim terms) the functional and taxonomy output.\n\n2) Extraction of taxonomic profile\n\nTo understand a microbial community, we must first understand which organisms are present along with their abundance. There are several approaches to microbial taxonomic profiling, but this workflow (Figure 2(b)) uses the marker gene approach. MetaPhlAn2 checks every read against a database of approximately one million clade-specific marker genes from nearly 17,000 reference genomes (bacterial, archaeal, viral, and eukaryotic)16. For this particular step, we use all reads, including rRNA since they are useful for taxonomic profiling. The outputs of MetaPhlAn2 are a SAM (sequence alignment map) file and a BIOM (Biological Observation Matrix) file, which both show the mapping of reads onto the reference database, and Community Profile tabular output which contains information regarding the taxa present and their relative abundance. This table includes information at all taxonomic levels, so to parse it out into each separate level we use the Format MetaPhlAn2 tool.\n\nWe use two different tools to visualize the taxonomic profiles. First, we use Krona17, which creates an interactive pie chart from the hierarchical taxonomic data. This chart is multi-layered for each taxonomic level and can be zoomed for viewing at each level. GraPhlAn18 is the other visualization tool, which creates a publication-ready circular representation of a phylogenetic tree based on the taxonomic results. We must first use export2graphlan to convert the MetaPhlAn2 results to a format that GraPhlAn can use.\n\n3) Extraction of functional information\n\nAfter characterizing the taxonomic profile of each sample, we must determine which genes are expressed and the biological processes involved. To perform functional profiling (Figure 2(c)), we use HUMAnN24, which is a pipeline to quickly and accurately determine the presence and abundance of functional gene families and pathways from metagenomic or metatranscriptomics data.\n\nFor identifying the functions expressed by the community, we filter out the rRNA sequences, due to their high noise levels and the compute time needed for their analysis. The output of SortMeRNA and the identified community profile from MetaPhlAn2 helps HUMAnN2 to focus on the known sequences for the identified organisms. Software tools such as ‘Renormalize’, ‘Unpack Pathway abundance to show gene families’, ‘Create gene level families file’, ‘Group abundances’ and text manipulation tools such as ‘Select lines’, ‘group columns’ and ‘sort columns’ have been introduced in the ASaiM-MT workflow. This generates normalized abundances of gene families, gene pathways and GO slim terms present in each sample.\n\n4) Combine taxonomic and functional information\n\nThe final step (Figure 2(d)) in this analysis is to determine which microorganisms are contributing to the profile of functions indicated by the expressed RNA sequences. HUMAnN2 partially answers this question by including taxa in its gene family and pathway outputs, but it does not include the taxa's abundance, only the functional abundance. We can fill in this missing information with the MetaPhlAn2 results using the Combine MetaPhlAn2 and HUMAnN2 Outputs tools. This produces a table of functional terms and their abundances with the corresponding genus and species abundances for the taxa which contribute to said function via their expressed RNA sequences. The abundances are reported in RPK values (reads per kilobase), calculated as the sum of the scores for all alignments for a gene family. These alignment scores are calculated according to the number of matches of a specific sequence to its reference genome and further normalized to account for multiple reference genome matches.\n\n\nResults\n\nTo demonstrate the use of the ASaiM-MT workflow, we analyzed a representative metatranscriptomic data set obtained from a microbial community within a thermophilic biogas reactor19 which digests municipal food waste and manure (Figure 3). The microbial community was sampled from the bioreactor and transferred to a rich medium containing lignocellulose from Norwegian Spruce and incubated at 65°C as an enrichment strategy. Triplicate mRNA samples were taken in a time series from 0 to 43 hours after inoculation. The mRNA was sequenced for paired-end reads (2 x 125 bp) on one lane of an Illumina HiSeq 3000. For the purpose of this study, we took only one time point (8hr). The paired FASTQ files (forward and reverse reads) were then subjected to the ASaiM-MT workflow (Figure 2).\n\nA 100 µl inoculum was collected from a lab-scale biogas reactor incubated at 55 °C and transferred to an anaerobic flask containing 10 g/L of cellulose. Triplicate mRNA samples were taken in a time series from 0 to 43 hours after inoculation. Metagenomic and metaproteomic sequencing was performed for all time points. For this tutorial we used one of the triplicates (T1A) in the 8 hours’ time point.\n\nThe ASaiM-MT workflow consists of four steps, i) preprocessing of the data, ii) extraction of community profile, iii) extraction of functional information, and iv) combining taxonomic and functional information. The data is preprocessed to make it compatible for MetaPhlAn2 (taxonomy) and HUMAnN2 (Function) annotation of the data.\n\nTo extract the taxonomic profile, the MetaPhlAn2 suite was run on the adapter trimmed interlaced files. The Community Profile output contained the information regarding the microbiome community present in the sample along with its relative abundance at different levels, i.e., Kingdom, Phylum, Class, Order, Family, Genus, Species, and Strain (Figure 4). For example - (k__Bacteria\|p__Firmicutes\|c__Clostridia\|o__Thermoanaerobacterales\|f__Thermodesulfobiaceae\|g__Coprothermobacter\|s__Coprothermobacter_proteolyticus) states that kingdom is bacteria, class is clostridia, belonging to order thermoanaerobacter, family of thermodesulfobiaceae, genus is copthermobacter and species is Comprothermobacter proteolyticus. As it is a cellulose-degrading consortium from anaerobic digestion, Coprothermobacter and Clostridium were expected to be identified for this dataset, demonstrating the accuracy of these tools.\n\nA tabular representation of MetaPhlAn2 community profile displaying the different levels of taxonomic classification and its relative abundance at that level.\n\nThe community profile is further processed using the Format MetaPhlAn2 tool which splits the MetaPhlAn2 output and categorizes them into various taxonomy levels (Kingdom, Phylum, Class, Order, Family, Genus, Species) with corresponding abundance values. Supplementary Figure S1 (Extended data20) shows genus level relative abundance values associated with genera present in the dataset. For visualization of the taxonomic profile, GraPhlAn and Krona (interactive) were used (Figure 5a and 5b).\n\na) The Krona output provides interactive representation of the community in the sample. In this case Coprothermobacter and Clostridium were the most abundant genera. b) The GraPhlAn outputs circular phylogenetic trees showing that Archaea and bacteria are present in the sample.\n\nThe HUMAnN2 suite of tools were used to extract functional information along with their relative abundance (RPK). HUMAnN2 provides 3 different outputs- Gene family and their abundance (Extended data: Supplementary Figure S2a20), pathways and their abundance (Extended data: Supplementary Figure S2b20) and pathway and their coverage (Extended data: Supplementary Figure S2c20).\n\nIn this workflow, the UniRef50 database was used to classify the gene family, but the users also have a choice to use UniRef90. Gene family abundance at the community level is stratified to show the contributions from known and unknown species. The gene family output shows total abundance value which is the sum total of individual species abundance values (reported as RPK values). Additionally, the tabular output also enlists the contribution of individual species to the gene family abundance (Extended data: Supplementary Figure S320). While there are some applications, e.g., strain profiling, where RPK units are superior to depth-normalized units, most of the time we need to renormalize our samples prior to downstream analysis. The gene families can be a long list of IDs and going through the gene families one by one to identify the interesting ones can be cumbersome and error prone. To help construct “the bigger picture”, we could identify and use categories of genes using the gene families. Gene Ontology (GO) analysis is widely used to reduce complexity and highlight biological processes in genome-wide expression studies. There is a dedicated tool called Group abundances of UniRef50 gene families obtained to gene ontology (GO) slim Terms, which groups and converts UniRef50 gene family abundances generated with HUMAnN2 into GO slim terms (Extended data: Supplementary Figure S420) as the name suggests.\n\nThe functional and taxonomic annotations from MetaPhlAn2 and HUMAnN2 are further normalized and combined to create a single tabular output.\n\n\nUse cases\n\nHere we provide a trimmed version of the biogas reactor dataset to demonstrate the use of the ASaiM-MT workflow in the tutorial available in the GTN.\n\nLink: https://training.galaxyproject.org/training-material/topics/metagenomics/tutorials/metatranscriptomics/tutorial.html\n\nTrimmed input: https://zenodo.org/record/3362849\n\nWorkflow: https://training.galaxyproject.org/training-material/topics/metagenomics/tutorials/metatranscriptomics/workflows/\n\n\nDiscussion\n\nThe ASaiM-MT workflow is made available in the Galaxy platform via ASaiM, enabling accessibility and flexibility for customization. There are a few tools that can be used alternatively or in addition to the existing tools. The ASaiM-MT workflow (as mentioned in the methods section) was tested to ensure that the workflow works on metatranscriptomics datasets. For details about default parameters used, we recommend visiting the metatranscriptomic tutorial available on the GTN material21.\n\nFor example, in ASaiM-MT, we map the UniRef 50 values to GO terms, but they can be also mapped to the MetaCyc reactions22, KEGG Orthogroups (KOs)23, Pfam domains24, EC categories25 and EggNOG (including COGs)26 using the HUMAnN2 regroup tool. A current limitation of the ASaiM-MT workflow is that it can process only one paired-end or single-end data at a time. In order to generate an input for statistical analysis, we have developed an additional post-processing workflow and Galaxy tool called MT2MQ (https://github.com/galaxyproteomics/tools-galaxyp/tree/master/tools/mt2mq), which integrates results from multiple outputs from the ASaiM-MT workflow. The MT2MQ workflow combines the gene abundance output from multiple samples or conditions, normalizes the values and makes it compatible with statistical tools such as metaQuantome tool13, which can be used for visualizing and interpreting results. Furthermore, we are in the process of developing tools that can help perform multi-omics studies by integrating results from the ASaiM-MT workflow to our existing metaproteomics workflows.\n\n\nConclusion\n\nASaiM-MT workflow is a robust and extensible Galaxy workflow which is now optimized and tested for metatranscriptomics data. The workflow consists of tested open-source tools in the area of RNA sequence analysis, such as SortMeRNA, MetaPhlAn2 and HUMAnN2. ASaiM-MT in Galaxy offers users a high-level control over their data and provides different analysis options. The GTN offers documentation and resources necessary for new users to gain mastery in using this workflow and associated tools for data analysis for their research projects. ASaiM-MT allows users to not only understand the taxonomy but also the functional composition and pathways expressed by the microbiome present in diverse microbial communities of interest.\n\n\nData availability\n\nZenodo: Training Data for “Metatranscriptomics analysis using microbiome RNASeq data”, http://doi.org/10.5281/zenodo.336284927.\n\nZenodo: Supplementary for ASaiM-MT: A validated and optimized ASaiM workflow for metatranscriptomics analysis within Galaxy framework, http://doi.org/10.5281/zenodo.434139120.\n\nThis project contains the following extended data:\n\n- Supplementary Figure S1: MetaPhlAn2 Genus-Level Abundance\n\n- Supplementary Figure S2a: HUMAnN2 Gene Family Abundance\n\n- Supplementary Figure S2b: HUMAnN2 Pathway Abundance\n\n- Supplementary Figure S2c: HUMAnN2 Pathway Coverage\n\n- Supplementary Figure S3: Uniref50 Gene Family output with abundance\n\n- Supplementary Figure S4: Conversion of Uniref 50 values to GO terms\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nSoftware availability\n\nSoftware available from: https://metagenomics.usegalaxy.eu/\n\nSource code available from: https://github.com/ASaiM/framework\n\nArchived source code at time of publication: http://doi.org/10.5281/zenodo.445562728.\n\nLicense: Apache 2 License\n\nDocker: https://quay.io/repository/bebatut/asaim-framework (command: docker pull quay.io/bebatut/asaim-framework).", "appendix": "Acknowledgements\n\nWe would like to thank European Galaxy team for the help in the support during Galaxy implementation. We would also like to thank Björn A. Grüning (University of Freiburg, Germany) for helping us during the implementation of the workflow in the European Galaxy platform.\n\n\nReferences\n\nMohajeri MH, Brummer RJM, Rastall RA, et al.: The role of the microbiome for human health: from basic science to clinical applications. Eur J Nutr. 2018; 57(Suppl 1): 1–14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBolyen E, Rideout JR, Dillon MR, et al.: Author Correction: Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. (Nature Biotechnology, (2019), 37, 8, (852-857). Nat Biotechnol. Nature Publishing Group; 2019; 37(9): 1091. PubMed Abstract \| Publisher Full Text\n\nSchloss PD, Westcott SL, Ryabin T, et al.: Introducing mothur: Open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol. 2009; 75(23): 7537–41. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFranzosa EA, McIver LJ, Rahnavard G, et al.: Species-level functional profiling of metagenomes and metatranscriptomes. Nat Methods. 2018; 15(11): 962–968. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTurnbaugh PJ, Gordon JI: An Invitation to the marriage of metagenomics and metabolomics. Cell. 2008; 134(5): 708–13. PubMed Abstract \| Publisher Full Text\n\nBashiardes S, Zilberman-Schapira G, Elinav E: Use of metatranscriptomics in microbiome research. Bioinform Biol Insights. 2016; 10: 19–25. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilmes P, Bond PL: Metaproteomics: Studying functional gene expression in microbial ecosystems. Trends Microbiol. Trends Microbiol; 2006; 14(2): 92–7. PubMed Abstract \| Publisher Full Text\n\nShakya M, Lo CC, Chain PSG: Advances and challenges in metatranscriptomic analysis. Front Genet. Frontiers Media S.A.; 2019; 10: 904. 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BMC Bioinformatics. 2011; 12: 385. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAsnicar F, Weingart G, Tickle TL, et al.: Compact graphical representation of phylogenetic data and metadata with GraPhlAn. PeerJ. 2015; 3: e1029. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKunath BJ, Delogu F, Naas AE, et al.: From proteins to polysaccharides: lifestyle and genetic evolution of Coprothermobacter proteolyticus. ISME J. 2019; 13(3): 603–617. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMehta S: Supplementary for ASaiM-MT: A validated and optimized ASaiM workflow for metatranscriptomics analysis within Galaxy framework. 2020. http://www.doi.org/10.5281/zenodo.4341391\n\nMetatranscriptomics analysis using microbiome RNA-seq data. [cited 2020 Nov 25]. Reference Source\n\nCaspi R, Billington R, Fulcher CA, et al.: The MetaCyc database of metabolic pathways and enzymes. Nucleic Acids Res. 2018; 46(D1): D633–D639. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKanehisa M, Sato Y, Kawashima M, et al.: KEGG as a reference resource for gene and protein annotation. Nucleic Acids Res. 2016; 44(D1): D457–62. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFinn RD, Bateman A, Clements J, et al.: Pfam: The protein families database. Nucleic Acids Res. Nucleic Acids Res; 2014; 42(Database issue): D222-30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDönertaş HM, Cuesta SM, Rahman SA, et al.: Characterising complex enzyme reaction data. PLoS One. 2016; 11(2): e0147952. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHuerta-Cepas J, Szklarczyk D, Heller D, et al.: EggNOG 5.0: A hierarchical, functionally and phylogenetically annotated orthology resource based on 5090 organisms and 2502 viruses. Nucleic Acids Res. 2019; 47(D1): D309–D314. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBatut B, Mehta S, Kumar P: Training Data for \"Metatranscriptomics analysis using microbiome RNASeq data\" [Data set]. Zenodo. 2019. http://www.doi.org/10.5281/zenodo.3362849\n\nBatut B, Grvl K, Hiltemann S, et al.: ASaiM/framework: ASaiM-MT release (Version ASaiM-MT). Zenodo. 2021. http://www.doi.org/10.5281/zenodo.4455627" }	[ { "id": "79463", "date": "18 Feb 2021", "name": "Caitlin Simopoulos", "expertise": [ "Reviewer Expertise Computational biology", "bioinformatics", "RNA-sequencing data pipeline development and analysis", "microbiomes" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors describe an optimized workflow for metatranscriptomic microbial community data named ASaiM-MT. ASaiM-MT is an extension of ASaiM, a workflow mostly focused on metagenomic data available via Galaxy. Using helpful diagrams, the authors describe how ASaiM-MT differs from the original ASaiM workflow, and detail which tools from the Galaxy platform are used. Finally, the authors use data from a biogas reactor study the pipeline and demonstrate results that can be expected using ASaiM-MT. I particularly enjoyed the fact that the article is accompanied by written training material for the workflow. I believe that tutorials and user-friendly tools expand the audience of traditionally “bioinformatician-only” tools. However, I have a few suggestions for the authors to strengthen their presentation of the metatranscriptomic workflow before publication.\n\nMajor comments:\nIn your discussion, you highlighted a major limitation of ASaiM-MT: the fact that it can only handle a single sample and does not complete comparative analysis. However, you also mention that you have developed post-processing tools for this reason. It might be good to highlight that ASaiM-MT is essential (in particular for data pre-processing and identification of functional and taxonomic information) to be completed before statistical analysis. In addition, is it possible to process data in ASaiM-MT in batches? This might also lessen the perceived limitation of the tool.\n\nSometimes it is not completely clear why ASaiM-MT is needed if ASaiM was developed for both meta-genomics and -transcriptomics. It is important to be specific and highlight the rationale for developing the ASaiM-MT workflow. An example: “For performing this action, the original ASaiM Shotgun workflow used the FASTQ-joiner to join the reads. However, in the ASaiM-MT version, we use the FASTQ interlacer. FASTQ interlacer joins the forward (/1) and the reverse reads (/2) using the sequence identifiers; sequences without designation will be named as single reads. The reason ASaiM-MT uses FASTQ-interlacer rather than FASTQ-joiner is because the joiner tool combines the forward and reverse read sequence together while the interlacer puts the forward and reverse read sequences in the same file while retaining the entity of each read along with an additional file with unpaired sequences.” Was this change made because FASTQ interlacer is more appropriate for RNA sequencing, or was it a change to improve on the original ASaiM workflow?\n\nThe abstract and introduction seem to emphasize the importance of studying the human microbiome and its connections to health and well-being, however, this manuscript uses a microbial community obtained from a biogas reactor. This is a very interesting community that warrants further research, but the introduction does not match the sample of interest. I suggest updating the introduction to focus less on the human microbiome and emphasize the importance of studying microbial communities in general. Alternatively, if you’d like to focus on human microbiomes, the example data used should reflect the human focus.\n\nIn your discussion, you say: “There are a few tools that can be used alternatively or in addition to the existing tools.” Does this mean that there are other tools like ASaiM-MT that can be used? Your introduction mainly listed tools that complete specific tasks and not an end to end workflow. If there are other workflow tools, a comparison should be highlighted in the discussion or introduction. What makes ASaiM-MT unique compared to other tools/workflows? What are its strengths? Is it more user-friendly than other tools? Expanding on this will help strengthen your conclusions about the tool itself.\n\nMinor comments:\nThere are sections that can read like a “grocery list” of software names (eg. second paragraph of the introduction). I understand why you are listing them, but it could be useful to emphasize that each of these tools performs a single task and need to be put together into a workflow for a complete experiment. These tools are also often missing citations.\n\nIt can be difficult to understand which words are actually software names. Is it possible to type them in a monospace font? Or to bold the names?\n\nAt times the “Methods” section can get confusing, particularly when MT is being compared to the original ASaiM. My suggestion is to include numbers or roman numerals in the “in between” steps as described in Figure 2. That way you can also reference them in the text. For example, in “a) Preprocessing” there would be i. Input files, ii. Quality Control, iii. Adapter Trimming… etc.. It might also be useful to include these headers (and numbers/letters) in your Methods section to let readers follow along.\n\n“PMID 30298254” is used instead of a citation\n\nIs the rationale for developing the new method (or application) clearly explained? Partly\n\nIs the description of the method technically sound? Partly\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "6496", "date": "19 Apr 2021", "name": "Subina Mehta", "role": "Author Response", "response": "We greatly appreciate the reviewer’s comments and suggestions. We have incorporated the required changes in the updated version of the manuscript. Major comments: 1. In your discussion, you highlighted a major limitation of ASaiM-MT: the fact that it can only handle a single sample and does not complete comparative analysis. However, you also mention that you have developed post-processing tools for this reason. It might be good to highlight that ASaiM-MT is essential (in particular for data pre-processing and identification of functional and taxonomic information) to be completed before statistical analysis. In addition, is it possible to process data in ASaiM-MT in batches? This might also lessen the perceived limitation of the tool. A: We would like to thank the reviewer for the comment. We have edited the abstract and mentioned that ASaiM-MT is essential for preprocessing and identification of taxonomy and functional information before performing comparative analysis. Also, Galaxy has a function of providing datasets as a collection rather than a single input which can be used to handle multiple datasets. In the Galaxy platform, we have other software tools such as MT2MQ and metaQuantome, which can perform comparative statistical analysis. 2. Sometimes it is not completely clear why ASaiM-MT is needed if ASaiM was developed for both meta-genomics and -transcriptomics. It is important to be specific and highlight the rationale for developing the ASaiM-MT workflow. An example: “For performing this action, the original ASaiM Shotgun workflow used the FASTQ-joiner to join the reads. However, in the ASaiM-MT version, we use the FASTQ interlacer. FASTQ interlacer joins the forward (/1) and the reverse reads (/2) using the sequence identifiers; sequences without designation will be named as single reads. The reason ASaiM-MT uses FASTQ-interlacer rather than FASTQ-joiner is because the joiner tool combines the forward and reverse read sequence together while the interlacer puts the forward and reverse read sequences in the same file while retaining the entity of each read along with an additional file with unpaired sequences.” Was this change made because FASTQ interlacer is more appropriate for RNA sequencing, or was it a change to improve on the original ASaiM workflow? A: Thank you for this comment. We replaced the FASTQ-joiner tool with the FASTQ interlacer to improve on the original workflow. The FASTQ interlacer tool maintains the integrity of reads by maintaining the forward and the reverse sequence identifiers, as compared to joining them into a single read file, as is done by FASTQ-joiner tool. 3. The abstract and introduction seem to emphasize the importance of studying the human microbiome and its connections to health and well-being, however, this manuscript uses a microbial community obtained from a biogas reactor. This is a very interesting community that warrants further research, but the introduction does not match the sample of interest. I suggest updating the introduction to focus less on the human microbiome and emphasize the importance of studying microbial communities in general. Alternatively, if you’d like to focus on human microbiomes, the example data used should reflect the human focus. A: We thank the reviewer for the comment and have edited the abstract and introduction to highlight the importance of microbiome research in ecology as well as clinical research. 4. In your discussion, you say: “There are a few tools that can be used alternatively or in addition to the existing tools.” Does this mean that there are other tools like ASaiM-MT that can be used? Your introduction mainly listed tools that complete specific tasks and not an end to end workflow. If there are other workflow tools, a comparison should be highlighted in the discussion or introduction. What makes ASaiM-MT unique compared to other tools/workflows? What are its strengths? Is it more user-friendly than other tools? Expanding on this will help strengthen your conclusions about the tool itself. A: We thank the reviewer for the comment. The tools mentioned in the discussion offer an alternative option to the tools that we have used in the AsaiM-MT workflow, especially if the users have a preference. However, although we consider these tools to be appropriate to metatranscriptomics research, we have not tested these tools. ASaiM-MT workflow, due to its availability in Galaxy, offers an user-friendly option to the existing command line tools. The ASaiM-MT workflow has been tested with different datasets to ensure its compatibility. There is a systematic documentation available for the usage of the workflow in the Galaxy Training Network (GTN). Users can also ask questions to developers and users via the Gitter channel, if needed. Minor comments: 1. There are sections that can read like a “grocery list” of software names (eg. second paragraph of the introduction). I understand why you are listing them, but it could be useful to emphasize that each of these tools performs a single task and need to be put together into a workflow for a complete experiment. These tools are also often missing citations. A: Thanks to the reviewer for the comment. The tools listed are alternatives to the existing tools in the workflow and have not been incorporated as a workflow. We have added citations to these tools. 2. It can be difficult to understand which words are actually software names. Is it possible to type them in a monospace font? Or to bold the names? A: Thanks to the reviewer for this comment. I have formatted the tool names by making them italic. 3. At times the “Methods” section can get confusing, particularly when MT is being compared to the original ASaiM. My suggestion is to include numbers or roman numerals in the “in between” steps as described in Figure 2. That way you can also reference them in the text. For example, in “a) Preprocessing” there would be i. Input files, ii. Quality Control, iii. Adapter Trimming… etc.. It might also be useful to include these headers (and numbers/letters) in your Methods section to let readers follow along. A: Thank you for the comment. I have reformatted the method section according to Figure 2. 4. “PMID 30298254” is used instead of a citation A: Thank you reviewer for pointing this out. I have made the required change." } ] } ]	1	https://f1000research.com/articles/10-103
https://f1000research.com/articles/9-1174/v1	28 Sep 20	{ "type": "Research Article", "title": "Dendritic neurons can perform linearly separable computations with low resolution synaptic weights", "authors": [ "Romain D. Cazé", "Marcel Stimberg", "Marcel Stimberg" ], "abstract": "In theory, neurons modelled as single layer perceptrons can implement all linearly separable computations. In practice, however, these computations may require arbitrarily precise synaptic weights. This is a strong constraint since both biological neurons and their artificial counterparts have to cope with limited precision. Here, we explore how non-linear processing in dendrites helps overcome this constraint. We start by finding a class of computations which requires increasing precision with the number of inputs in a Perceptron and show that it can be implemented without this constraint in a neuron with sub-linear dendritic subunits. Then, we complement this analytical study by a simulation of a biophysical neuron model with two passive dendrites and a soma, and show that it can implement this computation. This work demonstrates a new role of dendrites in neural computation: by distributing the computation across independent subunits, the same computation can be performed more efficiently with less precise tuning of the synaptic weights. This work not only offers new insight into the importance of dendrites for biological neurons, but also paves the way for new, more efficient architectures of artificial neuromorphic chips.", "keywords": [ "Dendrites", "computation", "linearly separable", "implementation" ], "content": "Introduction\n\nIn theoretical studies, scientists typically represent neurons as linear threshold units (LTU; summing up the weighted inputs and comparing the sum to a threshold)1. Multiple decades ago, theoreticians exactly delimited the computational capacities of LTU, also known as the Perceptron2. LTU cannot implement computations like the exclusive (the XOR), but they can implement all possible linearly separable computations, and a sufficiently large network of LTUs can approximate all possible computations.\n\nResearch in computer science determined the synaptic weights resolution necessary and sufficient to compute all linearly separable computation3,4, and they evolve exponentially with the number of inputs. Formally an LTU needs integer-valued weights so that \|wi\|≥12ne−4nβ2nlogn2−n, with β a constant, to be able to implement all linearly separable functions4. Consequently, an LTU with finite means cannot compute all linearly separable functions5.\n\nThis limitation poses multiple practical problems. In the nervous system, neurons need to maintain a large number of synapses or synapses with a large number of stable states. Neuromorphic chips illustrate the problem and synapses often occupy the majority of the space, up to ten times more than the space occupied by neurons themselves6. We demonstrate here that dendrites might be a way to cope with this challenge.\n\nDendrites are the receptive element of neurons where most of the synapses lay. They turn neurons into a multi-layer network7,8 because of their non-linear properties9,10. They enable neurons to compute linearly inseparable computation like the XOR or the feature binding problem11,12. We wonder here if dendrites can also decrease the synaptic resolution necessary to compute linearly separable computations.\n\nFirst, we investigate the three input variable computations implementable by an LTU with positive synaptic weights. Second, we extend the definition of one of these computations to an arbitrarily high number of inputs. Third, we implement this computation at a smaller cost in a neuron with two passive dendrites.\n\nThis work not only shows the usefulness of dendrites in the nervous system, but also paves the way for a new generation of more cost-efficient artificial neural networks and neuromorphic chips composed of dendritic neurons.\n\n\nMethods\n\nWe performed simulations in a spatially extended neuron model, consisting of a spherical soma (diameter 10 µm) and two cylindrical dendrites (length 400 µm and diameter 0.4 µm). The two dendrites are each divided into four compartments and connect to the soma at their extremity. In contrast to a point-neuron model, each compartment has a distinct membrane potential.\n\nThe membrane potential dynamics of the individual compartments follow the Hodgkin-Huxley formalism with:\n\n\n\nfor the somatic compartment and\n\n\n\nfor the dendritic compartments.\n\nHere, Vsoma and Vdend are the respective membrane potentials, Cm = 1µFcm−2 is the membrane capacitance, gL, g¯K, and g¯Na stand for the leak, the maximum potassium and sodium conductances respectively, and EL, EK, and ENa stand for the corresponding reversal potentials. The currents Ia represent the axial currents due to the membrane potential difference between connected compartments. The synaptic current Is arises from a synapse placed at the respective compartment. It is described by\n\n\n\nwith Es being the synaptic reversal potential and gs the synaptic conductance. This conductance jumps up instantaneously for each incoming spike and decays exponentially with time constant τs = 1 ms otherwise:\n\n\n\nThe dynamics of the gating variables n, m, and h are adapted from 13 and omitted here for brevity.\n\nThe parameter values are summarized in Table 1.\n\nNote that due to the absence of sodium and potassium channels in the dendrites, the dendrites are passive and cannot generate action potentials.\n\nAll simulations were performed with Brian 214. The code is available from: http://doi.org/10.5281/zenodo.402394315. It allows for reproducing the results presented in Figure 4, Figure 5 and Figure 6. To demonstrate that the details of the neuron model do not matter for the results presented here, the code can also be run with a simpler leaky integrate-and-fire model.\n\nWe first define as a reminder Boolean functions:\n\nDefinition 1.A Boolean function of n variables is a function on {0, 1}n into {0, 1}, where n is a positive integer.\n\nNote that a Boolean function can also be seen as a binary classification or a computation.\n\nA special class of Boolean functions, which are of particular relevance for neurons, are linearly separable computations:\n\nDefinition 2.f is a linearly separable computation of n variables if and only if there exists at least a vector w ∈ ℝnand a threshold Θ ∈ ℝ such that:\n\n\n\nwhere X ∈ {0, 1}nis the vector notation for the Boolean input variables.\n\nBinary neurons are one of the simplest possible neuron models and closely related to the functions described above: their inputs are binary variables, representing the activity of their input pathways, and their output is a single binary variable, representing whether the neuron is active or not. The standard model is a linear threshold unit (LTU), defined as follows:\n\nDefinition 3.An LTU has a set of n weightswi ∈ 𝒲and a threshold Θ ∈ 𝒯so that:\n\n\n\nwhere X = (X1, . . . , Xm) are the binary inputs to the neuron, and 𝒲 and 𝒯 are the possible values for synaptic weights and the threshold, respectively.\n\nThis definition is virtually identical to Def. 2, however, wi and Θ are no longer arbitrary real values, but chosen from a finite set of numbers depending on the implementation peculiarities and noise at which these value can be stabilised. It means that a neuron may not be able to implement all linearly separable functions. For instance, a neuron with non-negative weights can only compute positive linearly separable functions:\n\nDefinition 4.A threshold function f is positive if and only iff (X) ≥ f (Z) ∀(X , Z) ∈ {0, 1}nsuch that X ≥ Z (meaning that ∀i: xi ≥ zi).\n\nTo account for saturation occurring in dendrites, we introduce the sub-linear threshold unit (SLTU):\n\nDefinition 5.A SLTU with d dendrites and n inputs has a set of d × n weights wi,j∈ {0, 1} with n wi such that∑jwi,j=wi,d dendritic threshold θ ∈ 𝒯 and a threshold Θ ∈ 𝒯, such that:\n\n\n\nwith\n\n\n\nSuch a neuron model can compute all positive Boolean functions (see Def. 4) provided a sufficient number of dendrites and synapses11.\n\nWe used integer-valued and non-negative parameters both for the LTU and the SLTU without loss of generality. It allows to exactly determine the number of similar synapses necessary to implement a given computation. The code necessary to perform all simulations is available from: http://doi.org/10.5281/zenodo.402394315\n\n\nResults\n\nIn the following, we will look at computations that cannot be implemented in an LTU without using different strictly positive synaptic weights. The simplest case where this can occur is for three inputs. We list all such computations in Table 2. Of these computations, the first three (OR, AND/OR, and AND) do not require distinct synaptic weights; an LTU can implement these computations with the same weight for all synapses. This is not the case for the last two computations that we call the Dominant AND (D-AND) and the Dominant OR (D-OR): here, an LTU implementation needs to have one synaptic weight that is twice as big as the others (see Figure 1).\n\nImplementations of the D-AND where X0 is the dominant input. Squares represent synapses with their synaptic weight, and circles stand for transfer functions. Here, the transfer functions are threshold functions with the given value as their threshold. A: Implementation of the D-AND, note that X0 has twice the synaptic weight compare to the others. B: Implementation of the D-OR, note that we keep the same synaptic architecture and we only change the threshold of the transfer function.\n\nWe have named the first new computation the D-AND because it requires the activation of a dominant (D) input AND the activation of another input. The D-OR is the Boolean dual of the D-AND, i.e. obtained by replacing AND operations by OR, and vice versa. In this function, an activation of the dominant input alone triggers an output. The other way to trigger an output is to activate both X1 and X2 at the same time. These functions have a “dominant input“ – an input that is sufficient to make the output true (D-OR), respectively necessary to make the output true (D-AND).\n\nIn the present paper, we chose X0 as the dominant input, but we could have picked X1 or X2. There is nothing comparable in the other three computations which treat all inputs identically.\n\nAn LTU (Figure 1) implements the computation by using synaptic strength. We employed synaptic weights with integer values to reﬂect their finite precision. Even if synaptic weights can take real values, a finite precision means a finite number of values, where each integer represents the index of this value. The weight and threshold values to implement a function are obviously not unique. For example, we could multiply all the weights by 2 and set the threshold to 6 (D-AND), or 4 (D-OR) and obtain the same results. Here we always use the lowest possible integer values for synaptic weights, and the corresponding lowest possible threshold.\n\nThen, we wanted to implement the D-AND and D-OR computation in threshold units with non-linear dendritic subunits, as an abstraction of neurons with dendrites7.\n\nWe consider two types of non-linearities: a threshold function to model supra-linear summation; and a saturating function to model sub-linear summation (SLTU; see Methods). Both types of summation have been observed in dendrites. Dendritic spikes are a well-known example of supra-linear summation12, while sub-linear summation can be observed in completely passive dendrites due to the intrinsic saturation of synaptic conductance9.\n\nFigure 2 shows a minimal implementation of D-AND in a dendritic neuron, with a supra-linear summation in Figure 2A and a sub-linear summation (SLTU) in Figure 2B. In both cases, all synapses are of identical strength. However, note that in the supra-linear implementation in Figure 2A, the X0 input connects to both dendrites. Therefore, as we define an input’s synaptic weight as the total effect it has in the final summation stage (analogous to depolarisation measured in the soma of a neuron), we have to consider the weight of X0 as twice as high as the other inputs. Note that this makes this implementation similar to the implementation in an LTU (Figure 1A): the dominance of X0 is expressed by a stronger weight. This is not the case for the sub-linear implementation in Figure 2B, where the synaptic weights are identical. Here, the dominance of X0 is only expressed by its placement.\n\nSince the D-OR is the dual of the D-AND, its implementation also follows the same structure as the D-AND, but with the sub- and supra-linear implementations reversed. In this case, the supra-linear implementation uses placement to implement the dominance of X0 whereas the sub-linear implementation uses strength.\n\nIn real neurons, spiking and saturating dendrites both integrate inputs sub-linearly in a given range9,12. Therefore, we will focus on D-AND in the following section and further explore the potential of threshold units with SLTUs as an abstraction of dendritic integration in neurons.\n\nSquares represent synapses and circles represent transfer functions and their respective threshold/saturation values. Note that the final transfer functions (“somatic integration”) are always threshold units, whereas the transfer functions of the sub-units (“dendrites”) are threshold functions as an example for supra-linear summation in A, and saturating functions as examples of sub-linear summation in B. A: D-AND Implementation using supra-linear summation. Note that X0 has two synapses and therefore a synaptic weight of two. B: D-AND Implementation using sub-linear summation. Here, all inputs have identical synaptic weights.\n\nIn the previous section, we have limited our analysis to computations with three input variables. We will now extend the definition of the D-AND to an arbitrary number of input variables. As in the three-variable case, we will consider one input to be the dominant input (assumed to be X0, without loss of generality). This input has to be activated together with at least one of the non-dominant inputs. Formally, we therefore define fn(X ) as follows:\n\n\n\nwhere X is the n-dimensional input vector with elements X0. . . Xn.\n\nWe can implement this function in an LTU (Figure 3A), as well as in a SLTU (Figure 3B)\n\nSynaptic weights are in squares, and transfer functions are in circles. A: Minimal D-AND implementation in an LTU. Note that this implementation requires a synaptic weight that is n − 1 times bigger than the smallest weight. B: Implementation in an SLTU with sub-linear summation.\n\nA: A biophysical model with two dendrites and a soma (lines: dendrites, circle: soma). Coloured squares depicts synapses. The model has three equivalent synapses. One comes from an input encoding the shape (black square), and two situated on the other dendrite encode the colour (blue or green). B: Membrane voltage traces responding to either clustered (the two synapses activate on the same dendrite; aquamarine) or dispersed (the two synapses activate on distinct dendrites; black) synaptic activation at five distinct locations (dendrite where two synapses cluster at 350 µm, 250 µm, 150 µm, 50 µm, and soma). Note that at the point where two synapses cluster the clustered activation is larger than the dispersed activation, whereas in the soma the opposite is true.\n\nBottom: Eight somatic membrane responses depending on the active inputs. (gray: no synapse/only black/green/blue, green: black + green, blue: black + blue, aquamarine: green + blue, black: all inputs active). Note that this activity reproduces the truth table from Table 2.\n\nThe biophysical model receives input from three sources, where activation happens at regular intervals of 25 ms, with a random jitter of ±1 ms for each spike. We translate this activity into a binary pattern for each time bin of 25 ms. Bottom: The model’s membrane potential as measured in the soma. The response spikes implement the output of the D-AND computation as described in Table 2.\n\nIn the LTU implementation (Figure 3A), the D-AND of n variables requires that an input has a synaptic weight at least n − 1 times bigger than the other inputs, and the threshold has to grow accordingly.\n\nWe can summarise these observations in a proposition.\n\nProposition 1.To implement the D-AND, an LTU requires that an input has a synaptic weight n − 1 times bigger than the smallest synaptic weight.\n\nProof. The LTU must stay silent when X0 is not active, even if X1, X2, . . . , Xn−1 are active. Therefore w1+w2+...+wn−1 < Θ, thus Θ must be greater or equal than n × wmin with wmin the smallest synaptic weight.\n\nConversely, the output should be active as soon as X0 is co-active with any other input Xj (for j ≥ 1). So w0 + wmin ≥ Θ, this means w0 + wmin≥ n × wmin, thus w0 ≥ wmin(n − 1).\n\nIn contrast, Figure 3B provides a constructive proof that an SLTU can implement the D-AND with equal synaptic weights. In this implementation, the distinguishing feature of the dominant input is that it targets the second dendrite; the synaptic weights and the threshold do not have to change with the number of inputs. If one would only measure the response to single inputs at the “soma” (last stage of summation), the dominant input would be indistinguishable from the other inputs, despite its dramatically different importance.\n\nWe will see next how these insights transfer to a more realistic biophysical model.\n\nTo use an illustrative example, consider a predator of a certain shape (triggering input X0) and either coloured green or blue (inputs X1 and X2). For an animal that is a potential prey, a stimulus should only trigger a ﬂeeing response if the predator’s shape is accompanied by one of the correct colours. Neither its shape alone nor either or both of the colours without the shape should trigger it.\n\nFigure 4A presents a single neuron biophysical model implementing this function. One synapse comes from an input encoding for the shape of an object, and this synapse connects to the upper dendrite. The two others encode for two different colours, either green or blue, and they connect to the lower dendrite. All the synapses, taken individually, produce the exact same depolarisation at the soma because we place them at the same distance (350 µm) and give them the same maximal conductance (20 nS).\n\nWe first look at the sub-threshold behaviour by disabling the sodium channels (gNamax = 0). Figure 4B plots the voltage response at distinct locations on the lower dendrite, and finally in the soma. To probe the response we either activate a shape and one of the colours (black line Figure 4B), or both colours (aquamarine line). In both cases, we activate two synapses, but in the first case this activation is dispersed over the two dendrites whereas in the second case it is clustered on the lower dendrite. Despite activating the same number of synapses in both cases, and despite them all having the same strength, the depolarisation is markedly different. At the tip of the dendrite – where the synaptic stimulation takes place – a dispersed activation leads to a smaller depolarisation than the clustered activation. In the soma, we observe the opposite: the dispersed activation leads to a bigger depolarisation than the clustered activation. For the dispersed activation, we record a depolarisation of 9.3mV at the soma, whereas it is only 6.2mV for the clustered activation.\n\nWe can explain this observation by considering the synaptic driving force16. The synaptic current induced by the activation of the synapse depends on the distance between the membrane potential and the synapses’ reversal potential; when several inputs drive the membrane potential closer to the reversal potential (here 0mV, this driving force diminishes). The combined effect of multiple synaptic inputs is therefore smaller then what is expected from summing the individual effects. In other words, the dendrite performs sub-linear summation.\n\nNote that we could have expected a difference of up to 100% between the clustered and dispersed stimulation in the soma. In the actual simulation, the difference is closer to 50%, showing the non-trivial interaction taking place at the soma.\n\nThis means that even if all synapses have the same impact on the soma individually; even if we have a complete synaptic democracy17, the relative placement of the synapses strongly inﬂuences the somatic response.\n\nBased on the sub-threshold behaviour presented above, we will now show that we can implement the D-AND in a spiking neuron model. It is crucial to look at the supra-threshold behaviour as it is how the neuron communicates with the rest of the network. Moreover, back propagated action potentials might undermine the dendritic non-linearity disrupting the implementation18.\n\nWe can interpret Boolean inputs and outputs in different ways when we apply them to a biophysical spiking neuron model. Here, we will consider two interpretations. Firstly, we can think of an active input as corresponding to a continuous stimulation where the individual spikes arrive at random times, and of an active output as some spiking activity of the neuron (“rate interpretation”). Alternatively, we can think of active inputs as coincidentally arriving spikes within a certain time window, and accordingly of an active output as a single spike emitted in response (“spike interpretation”).\n\nWe present the model implementing the rate interpretation in Figure 5. We introduced this model earlier (Figure 4), except that it has now active sodium channels (gNamax = 650mScm–2). Each of its inputs (colours corresponding to the colours in Figure 4) activates 25 times according to a Poisson process.\n\nFigure 5 displays, from top to bottom, the model’s responses in five different situations:\n\nA single input activates, in this case the neuron remains silent. We obtain the same outcome whatever the chosen input.\n\nTwo dispersed inputs activate (black + green or black + blue), in these two scenarios the neuron fires.\n\nThe two clustered inputs (green + blue) activate, in this case the neuron remains silent as expected from our observation in Figure 4B.\n\nAll inputs activate, in this last case the neuron does not overly fire notably because of the refractory period.\n\nThis figure thus presents the response of the neuron model to all non-trivial cases, we have only omitted the case without any input activation (and therefore without any output activity).\n\nFinally, we show an implementation of the spike interpretation in Figure 6. This model is identical to the model shown previously (Figure 5), except for a slightly lower activation threshold of the sodium channels (VT = −55mV instead of VT = −50mV) to make it spike more easily. We discretize time into bins of 25 ms and decide randomly for each input whether it is active in each bin. If it is active, it activates at the beginning of the bin with a small temporal jitter (1 ms); inputs activating in the same bin therefore spike coincidentally. We can directly link these activations to Boolean variables that are either 0 (no spike) or 1 (spike). As Figure 6 shows, the neuron implements the D-AND and only spikes whenever the “shape input” (black) is active together with at least one of the “colour inputs” (blue and green).\n\nWe have shown that a biophysical model can implement the D-AND computation using a different strategy than the LTU. Each input has the same synaptic weight producing the same depolarisation at the soma. To distinguish between the inputs, the biophysical model uses location instead of strength: the dominant input (black) targets its own dendrite, while the two other inputs cluster on the same dendrite. With this strategy, the model can implement the D-AND. This implementation also works for two interpretations of the Boolean inputs and outputs – as elevated rates of spiking without temporal alignment, or as precisely timed coincident spikes.\n\n\nDiscussion\n\nIn the present work, we demonstrate a computation for which an LTU implementation requires a synaptic weight many times higher than the other. In an implementation using dendrites, however, all synaptic weights can remain equal.\n\nTwo observations support this conclusion: 1) We have proven that an LTU requires that an input has a synaptic weight n − 1 times larger than the smallest input to implement a computation slightly more complex than coincidence detection. 2) A biophysical model with two dendrites can implement the same computation with all synaptic weights being equal.\n\nThe first two Results sections look at a computation and one of its possible extension for an arbitrary number of uncorrelated inputs. Note that this extension of the D-AND from three to an arbitrary number of inputs is not the only possibility. We think that the extension we chose is a reasonable one and it allowed us to demonstrate an advantageous implementation in an SLTU compared to an LTU.\n\nNote also that our denomination of one input as “dominant” and the others as “non-dominant” is very related to the distinction between “driver” and “modulator” inputs19. This concept, where driver inputs are necessary to activate a neuron, but this activity can be modulated by other inputs, is ubiquitous in the sensory system. For example, neurons in the primary visual cortex require a stimulus in their classical receptive field. Stimuli in the so-called extra-classical receptive field cannot activate the neuron by themselves, but strongly modulate the response if presented together with a stimulus in the classical receptive field20. This distinction is not entirely applicable in our example, since the dominant input X0 is not sufficient to activate the neuron by itself. Nevertheless, both computations rely on making a distinction between synaptic inputs, which can be implemented by placing inputs on different dendrites as we have shown in this study.\n\nOur results also relate to a study21 where we made use of dendrites to implement another computation with relevance to biology, namely direction selectivity. In the earlier study, we demonstrated how dendrites make the implementation resilient to massive synaptic failure, and this past work shows that dendrites improve the computing robustness. The present work introduces another aspect of robustness. Thanks to the saturating dendrite, the weights of the \"non-dominant\" synapses do not have to be set precisely; the only requirement is that a single input is enough to saturate the dendrite.\n\nSeveral of our model properties fit with experimental observations. At least in some neurons, synapses at different positions tend to create the same depolarisation at the soma17. Furthermore, other experimental studies demonstrate examples of sublinear summation in dendrites9,8, notably in interneurons.\n\nOur findings might also have implications beyond neuroscience, in particular for engineering applications. Studies in computer science assert that even problems solvable by an LTU might not have a solution when weights have a limited precision5. Being able to implement functions with equal synaptic weights, or with a small range of weights as we have demonstrated for the implementations in an SLTU, therefore may be advantageous for computations with limited resources.\n\nIn conclusion, dendrites enable computations to be implemented in more efficient and robust ways than without. This is not only of interest to neuroscientists, but might also inspire the design of future artificial neural networks and compact neuromorphic chips.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nCode necessary to perform all simulations is available from: http://doi.org/10.5281/zenodo.402394315.\n\nLicense: MIT", "appendix": "Acknowledgements\n\nThis project started a long time ago within a team directed by Alain Destexhe and stems from multiple discussions with B. Teleńczuk. We also want to thank F. Danneville and C. Loyez for the stimulant discussions, Prof. A. Cappy for his remarks on the method, and M. Humphries for numerous valuable comments. Finally we want to thank A. Foust for making our scientific English easier to read and Ms Marini-Audouard for the proof-reading before submission.\n\nA previous version of this article is availabile on bioRxiv: https://doi.org/10.1101/2020.04.20.051342.\n\n\nReferences\n\nMcCulloch WS, Pitts W: A logical calculus of the ideas immanent in nervous activity. 1943. Bull Math Biol. 1990; 52(1–2): 99–115; discussion 73–97. PubMed Abstract \| Publisher Full Text\n\nMinsky M, Papert S: Perceptrons: an introduction to computational geometry. MIT Press, Cambridge Mass, 1957.\n\nMuroga S: Threshold logic and its applications. Wiley-Interscience, New York, 1971.\n\nHåstad J: On the Size of Weights for Threshold Gates. SIAM J Discrete Math. 1994; 7(3): 484–492. Publisher Full Text\n\nDraghici S: On the capabilities of neural networks using limited precision weights. Neural Netw. 2002; 15(3): 395–414. PubMed Abstract \| Publisher Full Text\n\nPfeil T, Potjans TC, Schrader S, et al.: Is a 4-Bit Synaptic Weight Resolution Enough? Constraints on Enabling Spike-Timing Dependent Plasticity in Neuromorphic Hardware. Front Neurosci. 2012; 6: 90. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPoirazi P, Brannon T, Mel BW: Pyramidal Neuron as Two-Layer Neural Network. Neuron. 2003; 37(6): 989–999. PubMed Abstract \| Publisher Full Text\n\nTzilivaki A, Kastellakis G, Poirazi P: Challenging the point neuron dogma: FS basket cells as 2-stage nonlinear integrators. Nat Commun. 2019; 10(1): 3664. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbrahamsson T, Cathala L, Matsui K, et al.: Thin Dendrites of Cerebellar Interneurons Confer Sublinear Synaptic Integration and a Gradient of Short-Term Plasticity. Neuron. 2012; 73(6): 1159–1172. PubMed Abstract \| Publisher Full Text\n\nPolsky A, Mel BW, Schiller J: Computational subunits in thin dendrites of pyramidal cells. Nat Neurosci. 2004; 7(6): 621–7. PubMed Abstract \| Publisher Full Text\n\nCazé RD, Humphries MD, Gutkin BS: Spiking and saturating dendrites differentially expand single neuron computation capacity. NIPS. 2012; 1070–1078. Reference Source\n\nGidon A, Zolnik TA, Fidzinski P, et al.: Dendritic action potentials and computation in human layer 2/3 cortical neurons. Science. 2020; 367(6473): 83–87. PubMed Abstract \| Publisher Full Text\n\nTraub RD, Miles R: Neuronal Networks of the Hippocampus. Cambridge University Press, May 1991. Publisher Full Text\n\nStimberg M, Brette R, Goodman DFM: Brian 2, an intuitive and efficient neural simulator. Elife. 2019; 8: e47314. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCazé RD, Stimberg M: Source code for publication \"Dendritic neuron can perform linearly separable computations with low resolution synaptic weights\" (Version 2020.09). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.4023943\n\nKoch C, Segev I: The role of single neurons in information processing. Nat Neurosci. 2000; 3 Suppl: 1171–1177. PubMed Abstract \| Publisher Full Text\n\nMagee JC, Cook EP: Somatic EPSP amplitude is independent of synapse location in hippocampal pyramidal neurons. Nat Neurosci. 2000; 3(9): 895–903. PubMed Abstract \| Publisher Full Text\n\nBehabadi BF, Mel BW: Mechanisms underlying subunit independence in pyramidal neuron dendrites. Proc Natl Acad Sci U S A. 2014; 111(1): 498–503. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSherman SM, Guillery RW: On the actions that one nerve cell can have on another: distinguishing “drivers” from “modulators”. Proc Natl Acad Sci U S A. 1998; 95(12): 7121–7126. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHubel DH, Wiesel TN: Receptive fields and functional architecture in two nonstriate visual areas (18 and 19) of the cat. J Neurophysiol. 1965; 28(2): 229–289. PubMed Abstract \| Publisher Full Text\n\nCazé RD, Jarvis S, Foust AJ, et al.: Dendrites Enable a Robust Mechanism for neuronal stimulus selectivity. Neural Comput. 2017; 29(9): 2511–2527. PubMed Abstract \| Publisher Full Text" }	[ { "id": "72166", "date": "19 Oct 2020", "name": "Sacha van Albada", "expertise": [ "Reviewer Expertise computational neuroscience" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper shows that having synaptic inputs onto separate dendrites can lower the resolution and spread of the synaptic weights necessary for performing computations. The authors illustrate this using both simple threshold units and biophysical neuron models, on the example of a particular function they call ‘dominant AND’. The work appears to be novel and correct, and is presented in a well-organized, comprehensible manner. It may have an impact not only in neuroscience but also on the implementation of artificial neural networks.\nMinor\nThe condition on the weights given in the second paragraph of the introduction was only derived under certain conditions (n>=8 a power of 2) and it is still only a minimal lower bound – some threshold functions might still require larger weights. It should thus not be presented as a general condition.\nThe work of Ujfalussy et al. seems relevant but is not cited.\nThe English can be improved in places. For instance in the 2nd paragraph of the Introduction: “synaptic weights resolution” → “synaptic weight resolution”, “compute all ...computation” → “perform all...computations”, “they evolve...with” → “the weights depend...on”. Also in other places, reword “compute computations”.\nIn the same paragraph “an LTU needs integer-valued weights” is a bit misleading. Of course the weights do not need to be integer-valued, they can just be taken to be integer-valued without loss of generality. In the last sentence of that paragraph, please clarify what you mean by “means”.\n“The dynamics of the gating variables...are adapted from...and omitted here for brevity”: Please clarify if the dynamics are taken to be identical to the dynamics in that reference (“adapted” suggests otherwise – in which case more details would be needed for completeness).\nIn Definition 2, I would write “at least one vector” instead of “at least a vector”.\nIn Definition 5, maybe instead of “n inputs” you want to write “n inputs onto each dendrite”, and in the sum over w_{i,j} indicate the limits of j (presumably 1 to n). “d dendritic threshold” → “d dendritic thresholds”. In the first equation of Def. 5, you use limits 0 and d, and 0 and n, corresponding to (d+1) * (n+1) weights, instead of d * n, so I suggest adjusting the limits. In the second equation of Def. 5, you use X on the left-hand side but Y on the right-hand side.\n“provided a sufficient number of dendrites and synapses” → “given a sufficient number of dendrites and synapses”\n“It allows to...” → “This allows one to...” or “This allows us to...” In the same sentence, please clarify what you mean by “similar synapses”.\nFig. 1 caption “compare to the others” → “compared to the others”\n“the other three computations which treat” → “the other three computations, which treat”\np. 6, top: The link between focusing on D-AND and the focus on sublinearity is unclear.\nFig. 4 caption: ‘coloured squares depicts’ → ‘coloured squares depict’\np. 7, top: ‘greater or equal than’ → ‘greater than or equal to’\np. 7: ‘If one would only measure’ → ‘If one only measured’\np. 8: ‘it has now active’ → ‘it now has active’\nPlease be clearer on the role of the active sodium channels in supporting the computation. The abstract speaks of passive dendrites.\nI would reword ‘the neuron does not overly fire notably’ to something like ‘the output firing rate remains moderate’.\nDiscussion: ‘many times higher than the other’ → ‘many times higher than the lowest of the other weights’ or similar\n‘one of its possible extension’ → ‘one of its possible extensions’\nThe sentence ‘In conclusion, dendrites...than without’ is not grammatically correct.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6287", "date": "22 Jan 2021", "name": "Romain Cazé", "role": "Author Response", "response": "The condition on the weights given in the second paragraph of the introduction was only derived under certain conditions (n>=8 a power of 2) and it is still only a minimal lower bound – some threshold functions might still require larger weights. It should thus not be presented as a general condition. We rewrote this part of the introduction in less technical term while improving its accuracy. The work of Ujfalussy et al. seems relevant but is not cited. We now cite a work their work in the introduction The dynamics of the gating variables...are adapted from...and omitted here for brevity”: Please clarify if the dynamics are taken to be identical to the dynamics in that reference (“adapted” suggests otherwise – in which case more details would be needed for completeness). We changed adapted to “identical, except for…”, detailing the one difference to the parameters used in the reference. In Definition 2, I would write “at least one vector” instead of “at least a vector”. In Definition 5, maybe instead of “n inputs” you want to write “n inputs onto each dendrite”, and in the sum over w_{i,j} indicate the limits of j (presumably 1 to n). “d dendritic threshold” → “d dendritic thresholds”. In the first equation of Def. 5, you use limits 0 and d, and 0 and n, corresponding to (d+1) * (n+1) weights, instead of d * n, so I suggest adjusting the limits. We implemented the changes and corrected our definition and kept these changes throughout the article. In the second equation of Def. 5, you use X on the left-hand side but Y on the right-hand side. These mistakes happened during the editorial process, we corrected them. Pp. 6, top: The link between focusing on D-AND and the focus on sublinearity is unclear. We upgraded figure two and three, and changed the text to make this link clearer. Finally, we corrected all the English mistakes underlined by the reviewer, clarified it, reworded the article to avoid compute/computation and we clarified what we meant by \"means\"." } ] }, { "id": "72167", "date": "23 Oct 2020", "name": "Fleur Zeldenrust", "expertise": [ "Reviewer Expertise Computational neuroscience" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper shows, by fundamental derivation and simulations, that a certain class of neural computations can be more efficiently and realistically be implemented by neurons with dendrites than without dendrites. This is relevant and timely, as there has recently been an increased interest in dendritic computation, both from neuroscience and from neuromorphic computing. It is also a logical follow-up of the first author’s previous work, as mentioned in the discussion. I really appreciate the way of abstracting the neural computations to Boolean functions, as they also did in their previous work, which makes the work fundamental and generally applicable. The general setup of the paper is clear and appropriate, and the derivations and simulations are done correctly and clearly. My main comments are minor and concern the presentation, concerning the language, some further explanations and a few citations. I will add them in detail below. Overall, if these minor edits are addressed I wholeheartedly recommend this paper.\n\nDetailed comments:\nP. 3\n“a sufficiently large network of LTUs can approximate all possible computations” I think it depends a bit on the structure etc whether this is true (see https://en.wikipedia.org/wiki/Universal_approximation_theorem). Anyway, could there be a citation to how exactly this is meant?\n\nThe formula you give for integer-valued weights does not give integer values? And n is not defined.\n\nThe use of ‘means’ (in ‘finite means’) is a bit confusing: I think you mean something like ‘resources’, but you can also read it as ‘averages’.\n\n“Neuromorphic chips illustrate the problem and synapses often occupy the majority of the space, up to ten times more than the space occupied by neurons themselves” – I don’t really understand this sentence. How to these chips illustrate the problem? I also find the ‘and’ not logical, how does this belong together? I also miss a bit a conclusion, a ‘so…..’\n\n“Dendrites are the receptive elementS of neurons where most of the receiving synapses lay.”\n\n“They enable neurons to compute linearly inseparable computationS”\n\n“First, we investigate the three input variable computations implementable by an LTU”. I miss a sentence before this (something like: “We address this question by looking at…”). Also, what does ‘the three computations’ refer to? Which three computations? If there are only three, please include a reference or an explanation.\n\n“Third, we implement this computation at a smaller cost in..” Please define cost, otherwise it is meaningless.\n\n“This work not only shows the usefulness…” – better something like ‘a possible role’ or ‘a possible function’\n\n“and connect to the soma at their extremity” at ONE extremity.\n\nMerge paragraphs (i.e. delete white line) after eq. 2 (…compartments)\n\nMerge paragraphs (i.e. delete white line) after eq. 4 (… brevity)\nP. 4\n“the code can also be run with a simpler leaky integrate-and-fire model.” Is that code included at the repository? If yes, say so explicitly, if not, delete this sentence.\n\n“peculiarities” replace with something like ‘the specific implementation’\n\nDefinition 4: X ≥Z: what does this mean for vectors? The norm? Please specify.\n\nDefinition 5: there are a few things unclear to me:\nθ is defined but not used. Shouldn’t that be in the definition of E?\n\nIf the sum of w over j (wi) and all weights are either 0 or 1, then as soon as one synapse Xj with a weight wij is 1, E is 1, is that correct?\n\nY is not defined\n\nI don’t see the sublinear summation here, can it be explained how this is sublinear summation? Also, a figure explaining this would really help, as it is one of the core definitions of the paper\n\n“computations that cannot be implemented in an LTU without using different strictly positive synaptic weights” ‘different’ is a bit confusing here (different from what?). Maybe ‘heterogeneous’?\n\n“We list all such computations in Table 2.” “Table 2. The five computations for n = 3 inputs”. I am a bit confused: do you mean all possible computations? How? I can fill in a random combination in that truth table, and then I have a computation you have not listed yet. So what do you mean? Could you explain this (or give a reference)?\nP. 5\n“An LTU (Figure 1) implements the computation” which computation does this refer to? Because you describe DOR and DAND before\n\n“Here we always use” add comma between here and we\n\nDo not start a paragraph with ‘Then’. Also, ‘Next’ might be better\n\n“sub-linear summation can be observed in completely passive dendrites due to the intrinsic saturation of synaptic conductance” Isn’t this just an effect of the driving force, as also mentioned later in the results?\n\n“Here, the dominance of X0 is only expressed by its placement.” Please explain more\n\nFigure 2B: please refer to an equation or other explicit definition of the ‘saturating functions’\nP. 6\n“…dendrites both integrate inputs sub-linearly in a given range. Therefore, we will focus on D-AND in the following section…” I don’t understand how the choice for D-AND follows from sublinear integration in dendrites.\n\nFigure 3B: please refer to an equation or other explicit definition of the ‘saturating functions’\n\nFigure 4\nThese EPSPs are huge! I understand why this is needed here (to get to the saturation), but it is not very biologically realistic. Could this be addressed in the discussion?\n\n“…Membrane voltage traces responding…” a voltage trace does not respond.\n\n“…locations (dendrite …” -> “ locations (at the dendrite…”\n\nThe locations of the arrows does not seem to quite correspond with the locations of measurements? At least the leftmost one should at the same position as the synapses (according to the text), not more towards the end of the dendrite, and I think the rightmost should be in the middle of the soma?\n\nIt would be nice if an example could be included to show that dendrites are needed, i.e. that it does not work without (saturating) dendrites\n\nFigure 5\nLegend: part explaining the top should not be bold.\n\nTop (activity of the inputs): for what input it this? 111?\n\nIt would be nice if an example could be included to show that dendrites are needed, i.e. that it does not work without (saturating) dendrites\n\nP. 7\nProof: there are two different fonts for Theta\n\nI’m not wild about the predator-prey analogy. I understand that you want a ‘real-world example’, but I think this one does not really apply, as something could not possibly be green and blue at the same time (and one would actually require some kind of mutual inhibition because of that). So I think this analogy only gives unnecessary confusion.\n\nMerge paragraphs (i.e. delete white line) ending with ‘…sublinear summation.’, ‘…at the soma.’ and ‘…response.’.\n\nReplace ; with , after individually.\n\nAdd ‘-‘ with back propagation, or make one word.\nP. 8\n\nMerge paragraphs (i.e. delete white line) ending with ‘…spike interpretation.’\n\nExplanation Figure 5: so how is a synapse activated? Also not clear from figure. Is it silent if it is not activated, and fires with 100 Hz if it is activated?\n\nMerge paragraphs (i.e. delete white line) ending with ‘…remain equal.’\n\n“The first two Results sections look at a computation and one of its possible extension”-> “In the first two sections in the Results we describe an example of a computation and one of its possible extentionS “ (a section does not look)\n\n“We think that the extension we chose is a reasonable one” Why? Please explain\n\n“Note also that…” do not start a paragraph that way.\n\n“Our study also relateS…”\n\nThe discussion reads at the moment a bit more like a collection of loose arguments than as a single section. Could it be rewritten a bit so it is a bit more of a fluent story?\n\nAnother discussion point that needs to be addressed in the discussion is plasticity and learning: whereas synaptic plasticity mechanisms that increase or decrease the synaptic weights are well known, plasticity mechanisms that change the locations of synapses much less, to my knowledge. So what would learning in such a system look like?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6288", "date": "22 Jan 2021", "name": "Romain Cazé", "role": "Author Response", "response": "We thank the reviewer for the thorough review and the constructive comments. We think we have addressed all the issues that have been raised, please find a detailed point-by-point reply below (original reviewer comments in italics): a sufficiently large network of LTUs can approximate all possible computations” I think it depends a bit on the structure etc whether this is true (see https://en.wikipedia.org/wiki/Universal_approximation_theorem). Anyway, could there be a citation to how exactly this is meant? We decided not to go into details about the structure of such networks and therefore used \"sufficiently large\" to both encompass width and depth of the network. We now cite the study by G. Cybenko which (to our knowledge) is one of the first demonstrations of the finding. The formula you give for integer-valued weights does not give integer values? And n is not defined. We have decided to no longer give the full formula, since its details are not relevant to our study (the formula could indeed give non-integer values, but it only established a lower bound). We now only state that the Hastad et al. “studied a computation implementable by an LTU only if its synaptic weight resolution grows exponentially with the number of inputs.” The use of ‘means’ (in ‘finite means’) is a bit confusing: I think you mean something like ‘resources’, but you can also read it as ‘averages’. We have removed the consufing use of the word ‘means’ and replaced it by ‘resources’. Neuromorphic chips illustrate the problem and synapses often occupy the majority of the space, up to ten times more than the space occupied by neurons themselves” – I don’t really understand this sentence. How to these chips illustrate the problem? I also find the ‘and’ not logical, how does this belong together? I also miss a bit a conclusion, a ‘so…..’ We rewrote the paragraph to clarify our point (a large number of resources in LTU-based neuromorphic chips have to be dedicated to synapses). “First, we investigate the three input variable computations implementable by an LTU”. I miss a sentence before this (something like: “We address this question by looking at…”). Also, what does ‘the three computations’ refer to? Which three computations? If there are only three, please include a reference or an explanation. We have rewritten the paragraph to make it clearer. The “three input variable computations” did not refer to three computations, but to computations of three input variables. We have rephrased this as “computations of three input variables” to avoid confusion. “Third, we implement this computation at a smaller cost in..” Please define cost, otherwise it is meaningless. We have precise this point by stating that the implementation uses “fewer synapses”. “This work not only shows the usefulness…” – better something like ‘a possible role’ or ‘a possible function’ We have changed this sentence to state that the work “proposes a new role”. “and connect to the soma at their extremity” at ONE extremity. We have change the sentence accordingly. Merge paragraphs (i.e. delete white line) after eq. 2 (…compartments) Merge paragraphs (i.e. delete white line) after eq. 4 (… brevity) We have merged the paragraphs and slightly changed the text surrounding the equations. P. 4 “the code can also be run with a simpler leaky integrate-and-fire model.” Is that code included at the repository? If yes, say so explicitly, if not, delete this sentence. Yes, The code in the repo can run both models. We have changed the text to make this point clearer. * “peculiarities” replace with something like ‘the specific implementation’* We have changed the text accordingly * Definition 4: X ≥Z: what does this mean for vectors? The norm? Please specify.* One vector is superior to another if all of its component are superior. This is specified as part of the definition (“meaning that for all i…”) Definition 5: there are a few things unclear to me: θ is defined but not used. Shouldn’t that be in the definition of E? Thank you for pointing this out. The θ was indeed meant to be the threshold in the function E, but for our purposes a fixed value of 1 is enough. We have therefore removed θ from the definition. If the sum of w over j (wi) and all weights are either 0 or 1, then as soon as one synapse Xj with a weight wij is 1, E is 1, is that correct? This is correct and we now mention this in the text. Y is not defined This was a printing error, Y is the argument of the function E (instead of X). We have corrected this. * I don’t see the sublinear summation here, can it be explained how this is sublinear summation? Also, a figure explaining this would really help, as it is one of the core definitions of the paper* The summation is sublinear due to the saturation of the function E, which is the simplest (and strongest) sublinear summation: E(1) = 1, but E(1 + 1) = E(1 + 1 + …) = 1 as well. The shape of the function is now more clearly depicted in Figures 2 and 3. * “computations that cannot be implemented in an LTU without using different strictly positive synaptic weights” ‘different’ is a bit confusing here (different from what?). Maybe ‘heterogeneous’?* We have rewritten this paragraph to make it clearer and now use ‘heterogeneous’ instead of ‘different’. “We list all such computations in Table 2.” “Table 2. The five computations for n = 3 inputs”. I am a bit confused: do you mean all possible computations? How? I can fill in a random combination in that truth table, and then I have a computation you have not listed yet. So what do you mean? Could you explain this (or give a reference)? The table only lists positive threshold functions (or, equivalently, functions implementable by an LTU with non-negative weights), not all random combinations have this property. We have explained this more clearly in the rewritten paragraph. P. 5 “An LTU (Figure 1) implements the computation” which computation does this refer to? Because you describe DOR and DAND before Our statement refers to both computations, we have rewritten the sentence to make this explicit. “sub-linear summation can be observed in completely passive dendrites due to the intrinsic saturation of synaptic conductance” Isn’t this just an effect of the driving force, as also mentioned later in the results? Yes, we have changed the sentence to refer to the driving force here as well. “Here, the dominance of X0 is only expressed by its placement.” Please explain more We have expanded the explanation of the mechanisms used by the SLTU vs. the LTU. Figure 2B: please refer to an equation or other explicit definition of the ‘saturating functions’ We now refer to the definition of the function E in Def. 5. P. 6 “…dendrites both integrate inputs sub-linearly in a given range. Therefore, we will focus on D-AND in the following section…” I don’t understand how the choice for D-AND follows from sublinear integration in dendrites. We agree that this sentence was confusing and have removed it. Figure 3B: please refer to an equation or other explicit definition of the ‘saturating functions’ We now refer to Def. 5 in the caption. Figure 4 These EPSPs are huge! I understand why this is needed here (to get to the saturation), but it is not very biologically realistic. Could this be addressed in the discussion? These EPSPs were measured in the dendrite. When they are measured in the soma they are around 10mV and therefore more biologically realistic. While our reorganized Figure 4 no longer shows the EPSP measured at the dendrite, we now mention this point in the discussion. The locations of the arrows does not seem to quite correspond with the locations of measurements? At least the leftmost one should at the same position as the synapses (according to the text), not more towards the end of the dendrite, and I think the rightmost should be in the middle of the soma? In our reorganized figure, the arrows are no longer needed. It would be nice if an example could be included to show that dendrites are needed, i.e. that it does not work without (saturating) dendrites The reorganized Figure 4 now shows linear summation (i.e., without saturation) as a gray dotted line. Figure 5 Legend: part explaining the top should not be bold. Top (activity of the inputs): for what input it this? 111? It would be nice if an example could be included to show that dendrites are needed, i.e. that it does not work without (saturating) dendrites We corrected the mistake in the caption and add a final sentence to explain what would happen in a point neuron: all the voltage responses (clustered or scattered) would be equal. P. 7 Proof: there are two different fonts for Theta Thank you for alerting us to this issue, we will watch out for it in the revision proof. I’m not wild about the predator-prey analogy. I understand that you want a ‘real-world example’, but I think this one does not really apply, as something could not possibly be green and blue at the same time (and one would actually require some kind of mutual inhibition because of that). So I think this analogy only gives unnecessary confusion. We agree and have removed the paragraph. P. 8 Explanation Figure 5: so how is a synapse activated? Also not clear from figure. Is it silent if it is not activated, and fires with 100 Hz if it is activated? Yes, an “active synapse” receives 25 spikes over 250ms (i.e. a 100Hz input) and “inactive synapses” do not receive any spikes. We now point this out in the Figure caption and changed the text to make it clearer. “We think that the extension we chose is a reasonable one” Why? Please explain We have removed this sentence and instead now include a sentence describing the features of the function that are preserved by extending it in the way we did. The discussion reads at the moment a bit more like a collection of loose arguments than as a single section. Could it be rewritten a bit so it is a bit more of a fluent story? We have rewritten parts of the Discussion to make it more fluent. Another discussion point that needs to be addressed in the discussion is plasticity and learning: whereas synaptic plasticity mechanisms that increase or decrease the synaptic weights are well known, plasticity mechanisms that change the locations of synapses much less, to my knowledge. So what would learning in such a system look like? We have added a paragraph discussing the issue of plasticity to the discussion. We thank the reviewer for alerting us to a number of typos (missing ‘s’ at the end of words, missing commas, etc.) and language issues which we have corrected in the revised manuscript." } ] }, { "id": "72169", "date": "27 Oct 2020", "name": "Alexandra Tzilivaki", "expertise": [ "Reviewer Expertise computational neuroscience", "dendrites" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this modelling work, Caze and Stimberg propose that dendrites can efficiently decrease the weight resolution required to perform linear separable functions, something that is difficult to achieve using an LTU. The code that generates the model/data shown in figures is available and can be executed smoothly. All in all, the paper is a good fit for the F1000 Research. The results have the potential to advance the implementation of ANNs and neuromorphic hardware. Thus, it applies both to neuroscientific and computer science community.\nSuggestions:\nThe authors should test/report the robustness of their results by performing a sensitivity analysis for specific parameters of the model. For example, they could vary the number of dendrites, apply small changes in capacitance (Cm).\n\nWhat would be effect of using negative synaptic weights in the LTU vs. the \"dendritic neuron\".\n\nThe term \"dendritic neuron\" is not ideal as it resembles dendritic cells, which are cells in the immune system. I would propose the use of a different term, e.g. \"Neuronal dendrites\" or \"Neurons with dendrites\".\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6289", "date": "22 Jan 2021", "name": "Romain Cazé", "role": "Author Response", "response": "The authors should test/report the robustness of their results by performing a sensitivity analysis for specific parameters of the model. For example, they could vary the number of dendrites, apply small changes in capacitance (Cm). The results we present do not depend on model details, the only mandatory feature is the sub-linear summation in the dendrite. To further emphasize this point, we have added an exploration of the synaptic weight parameter in Fig.4 and discuss how it shows the robustness of the approach. Also note that the simulation code we provide can reproduce the basic results with a simple integrate-and-fire neuron model, further showing the robustness of the results. What would be effect of using negative synaptic weights in the LTU vs. the \"dendritic neuron\". In general, introducing negative weights will require the weights of other synapses to be higher in order to compensate. Be aware that the D-AND function cannot be implemented (neither in the LTU nor in the SLTU) if one of the inputs only connects with a single synapse that has a negative weight. This is because for each input, there is at least one configuration of the other inputs where switching that specific input from 0 to 1 needs to switch the output result from 0 to 1. If it only connects with a negative weight, this is not possible. The term \"dendritic neuron\" is not ideal as it resembles dendritic cells, which are cells in the immune system. I would propose the use of a different term, e.g. \"Neuronal dendrites\" or \"Neurons with dendrites\". We have changed the title to “Neurons with dendrites” to avoid any possible confusion." } ] } ]	1	https://f1000research.com/articles/9-1174
https://f1000research.com/articles/10-295/v1	16 Apr 21	{ "type": "Data Note", "title": "CFM: a database of experimentally validated protocols for chemical compound-based direct reprogramming and transdifferentiation", "authors": [ "Alexey Sizykh", "Khalimat Murtazalieva", "Yulia Vyshkvorkina", "Alexey Stupnikov", "Yulia A. Medvedeva", "Alexey Sizykh", "Khalimat Murtazalieva", "Yulia Vyshkvorkina", "Alexey Stupnikov" ], "abstract": "Cell fate engineering technologies are critically important for basic and applied science, yet many protocols for direct cell conversions are still unstable, have a low yield and require improvement. There is an increasing need for a data aggregator containing a structured collection of protocols - preprocessed, verified, and represented in a standardized manner to facilitate their comparison, and providing a platform for the researchers to evaluate and improve the protocols.\n\nWe developed CFM (cell fate mastering), a database of experimentally validated protocols for chemical compound-based direct reprogramming and direct cell conversion. The current version of CFM contains 169 distinct protocols, 113 types of cell conversions, and 158 small molecules capable of inducing cell conversion. CFM allows stem cell biologists to compare and choose the best protocol with high efficiency and reliability for their needs. The protocol representation contains PubChem CIDs and Mechanisms Of Action (MOA) for chemicals, protocol duration, media , and yield with a comment on a measurement strategy. Ratings of the protocols and feedback from the community will help to promote high-quality and reproducible protocols. We are committed to a long-term database maintenance strategy. The database is currently available at https://cfm.mipt.ru}{cfm.mipt.ru", "keywords": [ "Cell Fate Engineering", "Chemical reprogramming", "Direct cell conversion", "Transdifferentiation" ], "content": "Introduction\n\nCell engineering technologies possess a tremendous potential for basic and applied science. Recent discoveries in this field pushed forward regenerative and personalized medicine, drug discovery, and toxicology. In the future, cell engineering technologies can enable regeneration of altered tissues and extend the human lifespan. Now they serve as a platform for drug testing and toxicology experiments. Furthermore, these approaches enable molecular mechanisms to be revealed that drive normal development and differentiation. Transcription factors (TFs) have been used to manipulate cell fate in the majority of published protocols.1 Low efficiency and safety concerns restrict the application of cell fate engineering approaches in clinical practice.2 Small molecules that target signaling pathways or regulate the epigenetic state offer powerful tools for refined manipulation of cell fate to the desired outcome.3 There is an increasing number of protocols that utilize small molecules in order to induce lineage differentiation or to facilitate transdifferentiation by increasing efficiency or by replacing genetic reprogramming factors.\n\nSmall-molecule-mediated approaches have more potential for clinical applications since:3\n\n1. The biological effects of small molecules are typically rapid, reversible, dose-dependent and predictable, allowing precise control over specific outcomes and precise resembling of target cell type;\n\n2. Compared with genetic interventions, the relative ease of handling and administration of small molecules make them more practical for further therapeutic development;\n\n3. Small molecules do not evoke safety concerns about possible genome integration in contradistinction to genetic interventions.\n\nChemical cell conversion protocols vary not only in chemicals used for reprogramming but also in media, making it difficult to choose the optimal protocol in practice. A database integrating cellular reprogramming protocol has been already published.4 Yet, the protocol representation is not standardized and the database has not been updated recently. Here emerges the need for aggregation of protocols in a standardized form that would contain all relevant and specific information from them. Also, aggregators should provide user-friendly access to enable expeditious retrieval of information.\n\nIn this work, we present a protocol aggregator, CFM (cell fate mastering), with a user-friendly interface that allows stem cell biologists to compare protocols, select the best one for their needs with high efficiency and reliability as well as leave feedback for their experience regarding the protocol for the research community. Additionally, we provide information about a potential mechanism of drug action and pathways involved in cell conversion. Data on molecular mechanisms affected by specific compounds could help to improve the protocol. We use expert opinions to develop an original standard for protocol representation, provide public ratings for protocols calculated based on users’ evaluation, and maintain a discussion forum. Protocol ratings as well as detailed feedback from the community will help to promote high-quality and reproducible protocols. Standardized and detailed protocol representation will facilitate development and validation of systematic computational approaches for cell fate engineering (e.g. DECODE5). The current version of CFM contains 169 distinct protocols, 113 types of cell conversions, and 158 small molecules. Summary statistics are available in Figure 1.\n\n(A) Number of types of cell conversions. (B) Number of cell type conversion pairs in CFM (the bar for ”other 106 cell conversions” represents an averaged number of a protocols for each other cell conversions type). (C) Frequencies of chemical compounds used in the protocols. (D) Frequencies of initial cell types used in protocols.\n\nWe are committed to a strong database maintenance strategy, which means:\n\n1. We manually check data in order to provide comprehensive information. Public rating is conducive to promoting reproducible protocols;\n\n2. We communicate with experimental biologists and permanently improve and extend the functionality of the database to meet their needs. Hence, we created a special feedback form where users can inform us what features they would like to add;\n\n3. We support protocol updating by users (after manual curation of the suggested protocol);\n\n4. We also plan to update the content of the database on a regular basis (at least bi-annually).\n\n\nMethods\n\nWe collected protocols from PubMed and Google Scholar using the keywords ‘direct reprogramming’ AND ‘chemicals OR small molecules’, ‘transdifferentiation’ AND ‘chemicals OR small molecules’, ’direct cell conversion’ AND ‘chemicals OR small molecules’. In this way, more than 1000 papers were obtained. Experts prefiltered papers based on the content of key sections (abstract, introduction, method) discarding irrelevant papers. We manually extracted information from each relevant article and added data about small molecules implicated in protocol from PubChem as a part of postprocessing. The overview of the whole workflow can be seen in Figure 2.\n\nThe database can be queried at cfm.mipt.ru/query page (Figure 3). By default, all protocols are listed. If no protocols match a query, the whole database is displayed (default view). Search is case-independent. The first column contains a link to protocol description in the cfm.mipt.ru/viewProtocol/<protocolId> format, where protocol ID is an inner identifier. The protocol description page contains article information (DOI, article title, and authors), source and target cell lines , initial cell culture description, the species from which the source cell line was obtained, the total protocol duration, the media in which the protocol was implemented, protocol yield (with comments on how it was calculated), chemicals (with their methods of effect is known), transcription factors, stress factors and growth factors used during the procedure and some comments on the protocol in general. Also, we provide a simple interface for rating protocols for registered participants. After curation, the rating will be available on the protocol page. All rated protocols are shown in a personal user account (cfm.mipt.ru/login). A researcher can add a protocol (and a published related paper) to the CFM database by submitting it through cfm.mipt.ru/add after the email is verified by a CFM administrator. Detailed information about verification can be found on the Add Protocol page.\n\nWe strongly believe that the development and maintenance of such a database will encourage researchers to keep improving protocols for cell conversion. Among all cell conversions, reprogramming protocol still dominates over trans-differentiation Figure 1. As it is clear from the Figure 1B and C, fibroblasts are widely used as an initial cell type in various protocols, while other cell types are underrepresented. Although fibroblasts might be an easy cell type to obtain in the clinic, other initial cell types such as blood cells could be also of use. As can be seen in Figure 1D, there is a small group of chemicals effective in several protocols, suggesting that they target a general mechanism. Possibly other chemicals from the same functional class, such as epigenetic drugs, should also be tested for their cell conversion potential. Further research on the regarding relations between cell conversions and chemicals, media, transcription factors, etc. should lead to the development of a computational framework that will facilitate acceleration of the search for conditions conducive to new cell conversions which are highly applicable in medicine (for example,6-8).\n\nWe perform all computations on the server side, so the user only needs to have a web browser to use our service. However, we recommend not to access the service from smartphones because our interface is not optimized for them.\n\n\nUse cases\n\nIn Figure 4, as well as on Figure 3, you can see examples of querying the database. New fields of search can be added using the green ADD button; unnecessary fields can be removed using the red DELETE button.\n\nWe calculate the similarity of molecules based on their SMILES. Retrieving Morgan fingerprints from them, we calculate the Tanimoto similarity and provide the result to the user. The interface can be seen in Figure 5.\n\n\nData availability\n\nAll data was gathered from open sources such as PubMed; links to the source articles can be found on protocol pages accessible from cfm.mipt.ru/query page.\n\nWe collected protocols from PubMed and Google Scholar, and downloaded SMILES for chemical compounds from PubChem. All data can be downloaded from our GitHub Repo.\n\n\nSoftware availability\n\nOur service is available at: http:cfm.mipt.ru\n\nSource code is avaliable from: https://github.com/lubitelpospat/CFM-source\n\nArchived source code as at time of publication: http://doi.org/10.5281/zenodo.4500125.9\n\nLicense: MIT", "appendix": "Acknowledgements\n\nThe authors are grateful to Valeria Tsvelaya for her help with the standardized description of the protocols. We are also grateful to the creators and maintainers of https://pubchem.ncbi.nlm.nih.gov.\n\n\nReferences\n\nFu Y, Xie X, Liu J: Chemical reprogramming and transdifferentiation. Curr Opin Genet Dev. 2017; 46: 104–113. 0959-437X. PubMed Abstract \| Publisher Full Text\n\nXu J, Li X, Deng H: Small molecule-induced cellular fate reprogramming: promising road leading to rome. Curr Opin Genet Dev. 2018; 52: 29–35. 0959-437X. PubMed Abstract \| Publisher Full Text\n\nZhang Y, et al.: Small molecules, big roles – the chemical manipulation of stem cell fate and somatic cell reprogramming. J Cell Sci. 2012; 125: 5609–5620. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShen Y, et al.: Rpdb: a database of experimentally verified cellular reprogramming records. Bioinformatics 2015; 31: 3237–3239. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNapolitano F, et al.: Automatic identification of small molecules that promote cell conversion and reprogramming. bioRxiv 2020.04.01.021089. 2020. Publisher Full Text\n\nZhu S, Wei W, Ding S: Chemical strategies for stem cell biology and regenerative medicine. Annu Rev Biomed Eng. 2011; 13: 73–90. PubMed Abstract \| Publisher Full Text\n\nXu A, Cheng L: Chemical transdifferentiation: closer to regenerative medicine. Front Med. 2016; 10(2): 152–165. PubMed Abstract \| Publisher Full Text\n\nTakeda Y, Harada Y, Yoshikawa T, et al.: Chemical compound-based direct reprogramming for future clinical applications. Biosci Rep. 2018; 38(3). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDmitrievich SA: Cfm-source. Feb 2021. Publisher Full Text" }	[ { "id": "83456", "date": "11 May 2021", "name": "Erdem B. Dashinimaev", "expertise": [ "Reviewer Expertise cell biology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis work is devoted to creating a database of direct reprogramming protocols for human and mammalian cells. As a researcher working in this field, I welcome such initiatives to streamline and make sense of the large amounts of data emerging in this area. The database created by the authors is quite handy, illustrative, and can be used by other researchers as a convenient resource. However, I have a comment/suggestion to improve the content. It would be nice if two more parameters were offered for each protocol - the number of citations of the original article and the number of citations of the original article in reviews. Why I think this would be useful. It is no secret that the reproducibility of many published methods (even in top scientific journals) is very low. In my opinion, the citation rate of any article (not in reviews) can serve as a good marker that the method is reproducible with a high probability.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] }, { "id": "88121", "date": "28 Jun 2021", "name": "Stephanie M. Willerth", "expertise": [ "Reviewer Expertise Cell reprogramming", "tissue engineering" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper details the CFM database that enables users to explore, compare and evaluate different methods for chemical reprogramming cells from one phenotype into another. Cellular reprogramming is becoming an increasingly popular technique used for both understanding biology and for potential applications in regenerative medicine. The purpose of this database is clear as it provides a standard template for methodologies and evaluation of the effectiveness of these protocols for easy comparison for researchers who wish to use these methods. It also allows the community to rate the protocols. The database focuses on the use of small molecules to reprogram as opposed to transcription factor mediated reprogramming.\n\nThe database contains links to the papers containing the original protocols and the amount of data presented varies depending on the protocol. The methods I looked at had not yet been rated by users. The datasets have a standard format, but the listed information is not present for all protocols.\nI would suggest writing out Cell Fate Mastering (CFM) in the title of the article so it is easier for readers to understand. The references could be expanded to add more on the use of small molecules for reprogramming. I would suggest using consistent capitalization in Figure 2 and improving the resolution of Figure 1, the font size for the numbers on the y-axis in Figure 1 could be increased to improve readability.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-295
https://f1000research.com/articles/10-291/v1	16 Apr 21	{ "type": "Brief Report", "title": "The feasibility of targeted test-trace-isolate for the control of SARS-CoV-2 variants", "authors": [ "William Bradshaw", "Jonathan Huggins", "Alun Lloyd", "Kevin Esvelt", "William Bradshaw", "Jonathan Huggins", "Alun Lloyd" ], "abstract": "The SARS-CoV-2 variant B.1.1.7 reportedly exhibits substantially higher transmission than the ancestral strain and may generate a major surge of cases before vaccines become widely available, while the P.1 and B.1.351 variants may be equally transmissible and also resist vaccines. All three variants can be sensitively detected by RT-PCR due to an otherwise rare del11288-11296 mutation in orf1ab; B.1.1.7 can also be detected using the common TaqPath kit. Testing, contact tracing, and isolation programs overwhelmed by SARS-CoV-2 could slow the spread of the new variants, which are still outnumbered by tracers in most countries. However, past failures and high rates of mistrust may lead health agencies to conclude that tracing is futile, dissuading them from redirecting existing tracers to focus on the new variants. Here we apply a branching-process model to estimate the effectiveness of implementing a variant-focused testing, contact tracing, and isolation strategy with realistic levels of performance. Our model indicates that bidirectional contact tracing can substantially slow the spread of SARS-CoV-2 variants even in regions where a large fraction of the population refuses to cooperate with contact tracers or to abide by quarantine and isolation requests.", "keywords": [ "epidemiology", "SARS-CoV-2", "COVID-19", "contact tracing", "bidirectional tracing", "backward tracing", "B.1.1.7", "test-trace-isolate" ], "content": "Introduction\n\nThe frequency of the B.1.1.7 variant of SARS-CoV-2 has grown rapidly from its initial detection in October 2020 to become the dominant strain in southeastern England by the start of 2021. Studies have estimated the new strain is between 40% and 80% more contagious1,2. The rapid exponential growth of B.1.1.7, now found in dozens of countries, risks another and potentially higher wave of COVID-19 cases prior to widespread vaccination. Meanwhile, early reports suggest that current vaccines3 and prior SARS-CoV-2 exposure4 may be less protective against the B.1.351 and P.1 variants now common in South Africa and Brazil.\n\nAll three variants share an otherwise rare del11288–11296 mutation in orf1ab that can be detected using a single RT-PCR reaction5; B.1.1.7 can also be distinguished with the TaqPath diagnostic test6, twenty million of which are manufactured weekly7. As such, existing COVID-19 testing infrastructure can be used to track the transmission of the new variants. Samples testing positive by other kits can be re-screened8 without an emergency use authorization.\n\nTest-trace-isolate (TTI) strategies have been widely used to mitigate the spread of SARS-CoV-29. Models by the present authors10 and others11 have found that incorporating backwards tracing to identify infector individuals could dramatically increase the efficacy of tracing programs. However, testing delays, mistrust, and low compliance have undermined the confidence of health authorities in the benefits of TTI12,13. Moreover, efficacy sharply decreases when caseloads are high14, as is true for SARS-CoV-2 – but not yet the variants – in many regions.\n\nGiven the current low prevalence of the variants in most jurisdictions and the ability to identify cases of the new variant using existing testing infrastructure, we hypothesized that TTI programs dedicated to controlling them could substantially reduce the harm inflicted prior to widespread vaccination of populations later in 2021, especially if vaccine reformulation is needed. Such programs could be enhanced through incorporation of bidirectional tracing10.\n\nHowever, the effectiveness of TTI strategies varies widely from region to region due to programmatic and population-level differences in variables such as the proportion of cases who share their contact history with contact tracers; the proportion who comply with quarantine and isolation requests; and the overall rate of tracing success. Given this variation, it is unclear whether tracing programs exhibiting realistic levels of performance could feasibly dampen the spread of the new variants.\n\nTo evaluate the potential benefits of applying targeted test-trace-isolate to control variants, we applied a branching-process model of COVID-19 contact tracing10 to estimate the change in the effective reproduction number achievable across a wide range of parameters.\n\n\nMethods\n\nIn our branching-process model10, each case generates a number of new cases drawn from a negative binomial distribution according to pre-specified incubation- and generation-time distributions (Table 1). Cases are identified and isolated based on symptoms alone or through contact tracing. Cases either comply with isolation requests or ignore them completely according to some fixed probability of compliance; cases that comply generate no further cases.\n\nSuccessful tracing depends on the identified case sharing their contact history with tracers, and on the contact in question taking place within the time window (measured in days pre-symptom onset for symptomatic cases, and days pre-identification for asymptomatic cases). Environmental transmission is assumed untraceable. Symptomatic cases require a positive test before initiating contact tracing.\n\nOutbreaks were initialized with 20 index cases to minimize stochastic extinction and designated as “controlled” if reaching extinction before reaching 10,000 cumulative cases. Effective reproduction numbers (Reff) were computed as the mean number of child cases produced per case10.\n\n\nResults\n\nTo investigate the potential for TTI to mitigate the spread of variants, we investigated the effective reproduction number achieved across a range of data-sharing and trace-success rates (Figure 1). To account for uncertainty in variant transmissibility, we explored outcomes for reproduction numbers between 1.2 and 2.0; these values assume that non-tracing interventions are already in place.\n\nNeighbor-averaged contour plots, showing Reff achieved by bidirectional manual contact tracing with a tracing window of (a) two or (b) six days pre-symptom onset, under different combinations of trace success probability (x-axis), rate of data sharing with manual contact tracers (y-axis), rate of compliance with isolation and quarantine (row) and base reproduction number (columns). Other disease parameters are specified in Table 1. Isolation of symptomatic cases is sufficient to reduce R even when no traces succeed and/or no cases share their data with contact tracers. “Trace success probability” refers to trace attempts that are not otherwise blocked by environmental transmission or refusal to share data.\n\nIn the absence of contact tracing, identification and isolation of symptomatic cases alone reduced Reff by 0.2 to 0.3 even when quarantine and isolation compliance was low (Figure 1, top rows). When identification and isolation left Reff substantially greater than 1 (when base R ≥ 1.4), moderate levels of tracing could have substantial effects.\n\nWhen contacts were traced up to two days prior to symptom onset, roughly 60–70% data sharing and trace success rates were required to achieve an Reff reduction of at least 0.1, relative to isolation alone. If the window was extended to six days pre-onset to enable more effective bidirectional tracing, roughly 45–55% data sharing and trace success was sufficient. Higher levels of data sharing and trace success could achieve substantially larger reductions: in many scenarios, 85% data sharing and trace success reduced Reff by >0.2 in the two-day case and >0.35 in the six-day case.\n\nDue to the exponential growth of uncontrolled epidemics, small reductions in Reff can have a large impact on the total number of downstream cases arising from a given index case over a given timespan. For example, under a simple geometric series approach, reducing Reff by 0.1 from a starting value between 1.2 and 2.0 reduces the total number of child cases after 10 generations by 37–43%; an Reff reduction of 0.2 results in a reduction in child cases of 61–66%. Given an average generation time of six days, 10 generations equates to roughly two months – enough time, given sufficient delay in the spread of the new variant, to vaccinate a substantial fraction of the population.\n\n\nDiscussion\n\nOur results suggest that regions with even moderately functional contact tracing programs focused on the new variants could substantially slow their spread. Given a two-day window for bidirectionally tracing contacts pre-symptom onset, our model predicts that a program with 70% trace success, 70% data sharing, and 70% compliance with isolation could achieve an Reff reduction of at least 0.1 relative to the no-tracing case. Given a six-day window for efficient bidirectional tracing, regions with just 50% data-sharing, trace success, and isolation compliance could achieve a reduction of 0.1.\n\nUnder simple assumptions, such a reduction would reduce the number of child cases produced in two months by roughly 40%, buying time for vaccination to immunize many more people. More effective tracing programs can achieve larger reductions. Higher rates of cooperation might be achieved through home visits by contact tracers; exoneration for anything discovered in the course of contact tracing13; and financial and other support of people in quarantine and isolation40. In principle, concentrating vaccination in communities experiencing out-of-control variant transmission could further impair viral spread and increase the sustainability of TTI for COVID-19 control.\n\nThese results assume a high availability of suitable diagnostic tests and a rapid and consistent testing turnaround. They also take no account of any medical, demographic, geospatial or behavioral variation between cases that could influence the spread of the new variants.\n\nOur results suggest that TTI programs could help slow the spread of more transmissible and vaccine-resistant variants in regions where they are currently rare, providing vital time for widespread vaccination. As TTI efficacy is limited at high caseloads14, these findings indicate that tracing programs should immediately prioritize controlling the new variants rather than less transmissible – but currently more widespread – ancestral strains.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nSoftware availability\n\nSource code available from: https://github.com/willbradshaw/covid-bidirectional-tracing.\n\nArchived source code as at time of publication: http://doi.org/10.5281/zenodo.427955741\n\nLicense: MIT License", "appendix": "Author contributions\n\n\n\nK.M.E. conceived the study. J.H.H. and A.L.L. identified a suitable model framework. W.J.B. designed and programmed the adapted model, advised by the other authors. W.J.B. ran all simulations and generated figures. All authors jointly wrote and edited the manuscript.\n\n\nAcknowledgments\n\nWe thank Aaron Bucher of the COVID-19 HPC Consortium and Amazon Web Services for granting us extra cloud compute credits.\n\n\nReferences\n\nVolz E, Mishra S, Chand M, et al.: Transmission of SARS-CoV-2 Lineage B.1.1.7 in England: Insights from linking epidemiological and genetic data. medRxiv. 2021. Publisher Full Text\n\nPublic Health England: Investigation of novel SARS-COV-2 variant – Variant of Concern 202012/01. 2020. Reference Source\n\nWu K, Werner AP, Moliva JI, et al.: mRNA-1273 vaccine induces neutralizing antibodies against spike mutants from global SARS-CoV-2 variants. bioRxiv. 2021; 2021.01.25.427948. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWibmer CK, Ayres F, Hermanus T, et al.: SARS-CoV-2 501Y.V2 escapes neutralization by South African COVID-19 donor plasma. bioRxiv. 2021; 2021.01.18.427166. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVogels C, Fauver J, Grubaugh N: Multiplexed RT-qPCR to screen for SARS-COV-2 B.1.1.7, B.1.351, and P.1 variants of concern v2 (protocols.io.brrhm536).protocols.io. 2021. Publisher Full Text\n\nBal A, Destras G, Gaymard A, et al.: Two-step strategy for the identification of SARS-CoV-2 variants co-occurring with spike deletion H69-V70, Lyon, France, August to December 2020. medRxiv. 2020. Publisher Full Text\n\nThermo Fisher representatives. 2021.\n\nLopez-Rincon A, Perez-Romero CA, Tonda A, et al.: Design of Specific Primer Set for Detection of B.1.1.7 SARS-CoV-2 Variant using Deep Learning. Cold Spring Harbor Laboratory. 2020; 2020.12.29.424715. Publisher Full Text\n\nKoetter P, Pelton M, Gonzalo J, et al.: Implementation and Process of a COVID-19 Contact Tracing Initiative: Leveraging Health Professional Students to Extend the Workforce During a Pandemic. Am J Infect Control. 2020; 48(12): 1451–1456. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBradshaw WJ, Alley EC, Huggins JH, et al.: Bidirectional contact tracing could dramatically improve COVID-19 control. Nat Commun. 2021; 12(1): 232. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEndo A, Centre for the Mathematical Modelling of Infectious Diseases (CMMID) COVID-19 Working Group, Leclerc QJ, et al.: Implication of backward contact tracing in the presence of overdispersed transmission in COVID-19 outbreak. medRxiv. 2020. Publisher Full Text\n\nLewis D: Why many countries failed at COVID contact-tracing - but some got it right. Nature. 2020; 588(7838): 384–387. PubMed Abstract \| Publisher Full Text\n\nMegnin-Viggars O, Carter P, Melendez-Torres GJ, et al.: Facilitators and barriers to engagement with contact tracing during infectious disease outbreaks: A rapid review of the evidence. PLoS One. 2020; 15(10): e0241473. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nContreras S, Dehning J, Loidolt M, et al.: The challenges of containing SARS-CoV-2 via test-trace-and-isolate. Nat Commun. 2021; 12(1): 378. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLavezzo E, Franchin E, Ciavarella C, et al.: Suppression of COVID-19 outbreak in the municipality of Vo’, Italy. medRxiv. 2020. Publisher Full Text\n\nGudbjartsson DF, Helgason A, Jonsson H, et al.: Spread of SARS-CoV-2 in the Icelandic Population. N Engl J Med. 2020; 382(24): 2302–2315. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMa S, Zhang J, Zeng M, et al.: Epidemiological parameters of coronavirus disease 2019: a pooled analysis of publicly reported individual data of 1155 cases from seven countries. medRxiv. 2020. Publisher Full Text\n\nRivett L, Sridhar S, Sparkes D, et al.: Screening of healthcare workers for SARS-CoV-2 highlights the role of asymptomatic carriage in COVID-19 transmission. eLife. 2020; 9: e58728. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMeyerowitz EA, Richterman A, Bogoch II, et al.: Towards an accurate and systematic characterisation of persistently asymptomatic infection with SARS-CoV-2. Lancet Infect Dis. 2020; S1473-3099(20)30837-9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZou L, Ruan F, Huang M, et al.: SARS-CoV-2 Viral Load in Upper Respiratory Specimens of Infected Patients. N Engl J Med. 2020; 382(12): 1177–1179. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHu Z, Song C, Xu C, et al.: Clinical characteristics of 24 asymptomatic infections with COVID-19 screened among close contacts in Nanjing, China. Sci China Life Sci. 2020; 63(5): 706–711. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi R, Pei S, Chen B, et al.: Substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (SARS-CoV-2). Science. 2020; 368(6490): 489–493. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChau NVV, Lam VT, Dung NT, et al.: The natural history and transmission potential of asymptomatic SARS-CoV-2 infection. medRxiv. 2020. Publisher Full Text\n\nCai J, Sun W, Huang J, et al.: Indirect Virus Transmission in Cluster of COVID-19 Cases, Wenzhou, China, 2020. Emerg Infect Dis. 2020; 26(6): 1343–1345. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFerretti L, Wymant C, Kendall M, et al.: Quantifying SARS-CoV-2 transmission suggests epidemic control with digital contact tracing. Science. 2020; 368(6491): eabb6936. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCheng HY, Jian SW, Liu DP, et al.: Contact Tracing Assessment of COVID-19 Transmission Dynamics in Taiwan and Risk at Different Exposure Periods Before and After Symptom Onset. JAMA Intern Med. 2020; 180(9): 1156–1163. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHe X, Lau EHY, Wu P, et al.: Temporal dynamics in viral shedding and transmissibility of COVID-19. Nat Med. 2020; 26(5): 672–675. PubMed Abstract \| Publisher Full Text\n\nJing QL, Liu MJ, Yuan J, et al.: Household Secondary Attack Rate of COVID-19 and Associated Determinants. medRxiv. 2020; 2020.04.11.20056010. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDu Z, Xu X, Wu Y, et al.: Serial Interval of COVID-19 among Publicly Reported Confirmed Cases. Emerg Infect Dis. 2020; 26(6): 1341–1343. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWei WE, Li Z, Chiew CJ, et al.: Presymptomatic Transmission of SARS-CoV-2 - Singapore, January 23-March 16, 2020. MMWR Morb Mortal Wkly Rep. 2020; 69(14): 411–415. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGanyani T, Kremer C, Chen D, et al.: Estimating the generation interval for coronavirus disease (COVID-19) based on symptom onset data, March 2020. Euro Surveill. 2020; 25(17): 2000257. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRussell TW, Golding N, Hellewell J, et al.: Reconstructing the global dynamics of under-ascertained COVID-19 cases and infections. medRxiv. 2020. Publisher Full Text\n\nZhao J, Yuan Q, Wang H, et al.: Antibody responses to SARS-CoV-2 in patients with novel coronavirus disease 2019. Clin Infect Dis. 2020; 71(16): 2027–2034. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWhitman JD, Hiatt J, Mowery CT, et al.: Test performance evaluation of SARS-CoV-2 serological assays. medRxiv. 2020; 2020.04.25.20074856. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEndo A; Centre for the Mathematical Modelling of Infectious Diseases COVID-19 Working Group,Abbott S, et al.: Estimating the overdispersion in COVID-19 transmission using outbreak sizes outside China [version 3; peer review: 2 approved]. Wellcome Open Res. 2020; 5: 67. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLauer SA, Grantz KH, Bi Q, et al.: The incubation period of 2019-nCoV from publicly reported confirmed cases: estimation and application. medRxiv. 2020. Publisher Full Text\n\nGriffin J, Casey M, Collins Á, et al.: Rapid review of available evidence on the serial interval and generation time of COVID-19. BMJ Open. 2020; 10(11): e040263. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHellewell J, Abbott S, Gimma A, et al.: Feasibility of controlling COVID-19 outbreaks by isolation of cases and contacts. Lancet Glob Health. 2020; 8(4): e488–e496. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcCann L, Allan W, Read P, et al.: Contact tracing using provider referral: how difficult is it? Sex Health. 2013; 10(5): 472–473. PubMed Abstract \| Publisher Full Text\n\nBodas M, Peleg K: Self-Isolation Compliance In The COVID-19 Era Influenced By Compensation: Findings From A Recent Survey In Israel. Health Aff (Millwood). 2020; 39(6): 936–941. PubMed Abstract \| Publisher Full Text\n\nBradshaw W: willbradshaw/covid-bidirectional-tracing:v1.0 (Version v1.0). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.4279557" }	[ { "id": "83641", "date": "04 May 2021", "name": "Akira Endo", "expertise": [ "Reviewer Expertise Infectious disease modelling" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study considers the effectiveness of contact tracing focused on variants in reducing the reproduction number. Focusing contact tracing efforts on variants is an interesting approach and may be relevant to the current situation of variant circulations worldwide. The model and the analysis themselves seem well constructed and implemented. However, the authors’ analysis only focuses on a single variant essentially, and does not account for some important aspects that need to be considered to estimate the effect of real-world contact tracing in the presence of multiple variants. As a result, I am not sure if this study provides new insights that are distinct from existing studies on contact tracing for a single-pathogen outbreak. In addition, it should be noted that given a fixed capacity for contact tracing, the reduction in the reproduction number would not be permanent if the outbreak continues to grow. I believe these issues, along with other comments detailed below, need to be addressed for this study to be truly of epidemiological and public health interest.\nMajor comments:\nPlease clarify how this study is distinct from existing studies on contact tracing considering a single-pathogen outbreak (including the authors’ own study cited here).\n\nThere seems to be a mismatch between the study motivation/context and the modelling approach. One of the points the authors are trying to make is that the contact tracing efforts should be focused on variants because they are of more epidemiological importance (due to potentially higher transmission or immunoescape). I do not disagree with this point, but there are several major issues regarding how it was handled in the manuscript.\n\nThe reproduction number R is used as an objective variable to measure the effect of contact tracing. This is useful to connect interventions and the dynamic evolution of the epidemic, but essentially assumes that the same level of tracing can continue everywhere long-term, regardless of the epidemic size. This is obviously not true as the authors also state in the manuscript. In conditions where R is above 1, transmission of variants would continue and overwhelms the tracing capacity at some point, pushing R back to the original value eventually. Focusing on R may be useful in identifying conditions required to control the outbreak (i.e. R<1), but it is unrealistic to consider that the tracing can keep R lower than the original value in a long term if the resulting value exceeds 1.\n\nVariants are no longer minor in many places now (see for example: https://covid.cdc.gov/covid-data-tracker/#variant-proportions), and I am not sure how much this assumption of ‘minor variants’ is relevant to the actual situation. Moreover, even in places where the variants are still minor, if the (effective) transmissibility of the variants is higher than the existing virus, they would rapidly replace the existing viruses, potentially in a few weeks/months. Exclusion of existing strains. The main argument regarding the tracing capacity is that the variants account for a small proportion of cases and thus can be handled if tracing focuses on these variants. However, even if such focused intervention is possible by tests that can distinguish variants, existing non-variant viruses may continue spreading if their R is above 1. Although such a situation may still have some benefit, e.g. if preventing the spread of immunoescaping variants would ensure the success of the vaccination program, such contexts should be clarified and discussed.\n\nCost and capacity. As discussed above, contact tracing would work as estimated here only until the capacity is reached. However, I feel efforts associated with tracing is not seriously considered in the analysis. For example, if all contacts of cases within the tracing period are traced, extending the tracing period from 2 days to 6 days would incur substantial additional effort for tracing. I believe it is important to discuss to what extent contact tracing might be sustainable for each setting because the presented results become invalid once the capacity is reached.\n\nGiven the points above, I would recommend the authors reconsider what outcome measure to use and how to present them; e.g. consideration of the growth of \"non-targeted\" viruses, conditions required to keep R below 1, whether tracing can “buy time” until achieving a sufficient level of vaccination before reaching the capacity, optimising the intensity of other NPIs (e.g. lockdowns) in the presence of contact tracing, etc., such that the results are relevant to what may actually happen.\nThe Introduction looks lightweight and lacking necessary details or contexts. There are a lot of concepts that may not be familiar enough to every reader but are not sufficiently explained (e.g. TTI, backward contact tracing, bidirectional tracing, why TaqPath test can distinguish B.1.1.7… etc.) and thus may require a succinct clarification. Please also note that this paper may be read in 20 years from now, when the reader may not have the same level of recognition of the current situation. In this light, for example, I feel the first paragraph of Introduction may sound a bit abrupt to the reader who is less aware of the overall timeline of the pandemic. Also see some of the specific comments in the Minor comments section.\n\nThe Methods section is too simple and does not contain sufficient information for the reader to comprehend the overall structure of the analysis. Although it does not need to contain every technical detail of the model and analysis as the supplementary methods can be found in the repository (but please include a link and description in the paper so that the reader can easily find it), I feel more information from the supplementary methods should be extracted and summarised in the main text. For example, from the current Methods section I cannot interpret how the course of transmission was characterised, what is the assumed procedure of tracing (Is it always bidirectional tracing? I feel 2-day window is too short for backward tracing), how environmental transmission was assumed to work, how R was calculated, etc.\n\nI believe additional sensitivity analysis would be necessary. For example, the overdispersion parameter (0.11 used in the current analysis) is estimated to be slightly higher (0.3-0.5) in some studies where interventions were in place (Adam et al., 20201). As the authors assume that interventions may be affecting R during contact tracing, possible changes in overdispersion should also be considered. Delay from secondary transmission to quarantine of contacts (defined as a sum of various delay distribution) would also affect the effectiveness of contact tracing in a nontrivial manner.\n\nIs the effect of vaccines not considered, although as in Introduction it was one of the major motivation for considering controlling variants? Vaccines may affect different viruses similarly or differently, depending on the type of variants.\n\nSupplementary Methods, “Identified contacts are quarantined, …isolated, tested, and traced as described above”: what is the difference between quarantining and isolation of traced contacts? Does this mean all traced contacts of a case are put under quarantine regardless of their true infection status, but only tested if they are symptomatic (which changes the label from quarantine to isolation)? If so, it is expected that as the epidemic grows there would be a substantial number of quarantined individuals, and at some point this might be impossible (e.g. due to depletion of essential workers) and the Reff control could collapse.\n\nMinor comments:\nThroughout: please spell out acronyms at their first appearance, including SARS-CoV-2 and COVID-19.\n\nIntroduction, protection against B.1.351 and P.1: now the evidence is not limited to in-vitro studies (e.g. Madhi et al., 20212 and Kustin et al., 20213). Please update and include clinical findings. Also summarise what we know about protection against B.1.1.7.\n\n“All three variants share…; B.1.1.7 can also be…”: I would suggest that the authors first describe B.1.1.7 that can be detected by TaqPath tests (with some more background context, as this is primarily happening in UK and not necessarily recognized by the wider audience) and then go on to a discussion of potential detectability of other variants (because this is only a hypothetical scenario so far in my understanding, as opposed to detection of B.1.1.7). Also, would there be any data on the rollout of these variant-distinguishable tests worldwide?\n\n“Samples testing positive…”: This needs more context. Why is authorisation going to be an issue and why can re-screening bypass it?\n\n“as is true for SARS-CoV-2 – but not yet the variants – in many regions”: I feel this is unclear. TTI capacity would be overwhelmed when the overall caseloads are high, even if the variants account for a very small fraction of them. It should be made clear if this indicates contact tracing would only target variants distinguished by the (variant-specific) tests.\n\nMethod, “child cases” may be interpreted as cases that are children. Secondary transmissions?\n\nResults, “In the absence of contact tracing, identification and isolation of symptomatic cases alone reduced Reff by 0.2 to 0.3…”: I couldn’t read this from the top rows of Figure 1. This may correspond to 0% of cases sharing data or 0% trace success probability, but Reff for such a scenario cannot be read from the figure because there is no colour scales or numbers.\n\n“When identification and isolation…substantial effects.”: I am not sure how “moderate levels” and “substantial effects” are defined.\n\n“Due to the exponential growth of uncontrolled epidemics…over a given timespan”: As stated above, this is only the case if contact tracing can continue without hitting the capacity. If R goes back to the original level after tracing is overwhelmed, there may be only a marginal difference in the final epidemic size.\n\nDiscussion, “Higher rates of cooperation…quarantine and isolation”: related to the first major comment, these efforts would make tracing more effective but require a substantial amount of effort and cost, and warrant discussion.\n\nPlease update references. Many of the preprints cited here have now been published in peer-reviewed journals, which might include more up-to-date information.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "84410", "date": "14 May 2021", "name": "Tim C. D. Lucas", "expertise": [ "Reviewer Expertise Epidemiology. Previous research on contact tracing for SARS-CoV-2." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this study the authors use established and previously published models of contact tracing to examine whether targeted test and trace systems could suppress novel variants. The premise is sound; contact tracing scales poorly, so while it is not necessarily effective at control SARS-CoV-2 at large once national prevalence is high, the numbers of certain variants are still low in a number of countries and therefore contact tracing might be able to control those new variants as they are seeded into a country. Whether this approach would work or not is not trivially obvious and so this study is asking an important question with policy implications globally.\nThe analytical approach taken is quite simple in that the authors assume (and back up with some literature) that the variants can be identified easily and that therefore contact tracing of a new variant can continue without any reference to the dominant variant.\n\nComments:\nMost of my comments relate to this assumption that contact tracing of new variants can be modelled by simply ignoring the dominant variant.\n\nFirst, I would like to see this assumption explicitly stated in the methods just to make it completely clear to the reader.\n\nThere are a number of further considerations with this assumption that I think should be discussed.\n\nGiven the high rate of vaccination and previous infection with the original SARS-CoV-2 strain, many countries are now in a state where immunity cannot be ignored. This is all handled by Reff, but I think it needs to be mentioned that Reff is combining NPIs, immunity or partial immunity from vaccination (depending on whether there's vaccine escape in the variant) and partial immunity from previous infection with other strains.\n\nThe authors state that new variants can be detected with RT-PCR and TaqPath. However, does this extra step create no extra delay in the process? I imagine this would depend on the specific organisation but might be worth considering and mentioning.\n\nFurthermore, is this identification of variants 100% accurate? The false negative rate (someone is infected with a new variant but the test says they are infected with the original variant) can be just included as part of the test sensitivity and I wouldn't be surprised if the difference is fairly small. More worrying for me is the false positive rate (someone is infected with the original variant but the tests says they are infected with a new variant). This is important because the rationale for the study relies entirely on the fact that there are not many cases with the new variant in a country but if, say, the false positive rate (as defined above) is even 1% then the large number of original variant cases in a country will quickly lead to the targeted test-trace-isolate system being swamped. This effect will obviously vary with the prevalence of original variant SARS-CoV-2.\n\nI only know the literature for the UK, but even the lowest compliance rates used here are much higher than those measured (I wouldn't be surprised if some countries have much high compliance rates though). I am taking my values from the reference below (Smith et al., 20201), but there might be more up-to-date surveys in the UK and I don't know at all about other countries.\n\nFrom self-reported behaviour (past behaviour, not intentions) in the UK, about 12% of people with symptoms requested a test. This relates to the 50% of symptomatic cases identified without tracing parameter. Some details of how you selected 50% from ref 32 would be useful, as the values in that paper range from 5% to 100% depending on the country and time. In the UK, of those contacted by track and trace, 11% of people fully complied with 2 weeks self isolation (this relates to the 50%-90% comply with isolation parameter). So at the very least I think it might be useful to state that these values might be quite optimistic in some settings.\n\nFinally, a minor and subjective point, but it might be useful to present Figure 1 with a diverging colour palette that clearly distinguishes Reff < 1 and Reff > 1.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-291
https://f1000research.com/articles/10-100/v1	11 Feb 21	{ "type": "Research Article", "title": "Top health research funders’ guidance on selecting journals for funded research", "authors": [ "Larissa Shamseer", "Kelly D. Cobey", "Matthew J. Page", "Jamie C. Brehaut", "Jeremy M. Grimshaw", "Sharon E. Straus", "Lesley A. Stewart", "David Moher", "Kelly D. Cobey", "Matthew J. Page", "Jamie C. Brehaut", "Jeremy M. Grimshaw", "Sharon E. Straus", "Lesley A. Stewart", "David Moher" ], "abstract": "Background: Funded health research is being published in journals that many regard as “predatory”, deceptive, and non-credible. We do not currently know whether funders provide guidance on how to select a journal in which to publish funded health research. Methods: We identified the largest 46 philanthropic, public, development assistance, public-private partnership, and multilateral funders of health research by expenditure, globally as well as four public funders from lower-middle income countries, from the list at https://healthresearchfunders.org. One of us identified guidance on disseminating funded research from each funders’ website (August/September 2017), then extracted information about selecting journals, which was verified by another assessor. Discrepancies were resolved by discussion. Results were summarized descriptively. This research used publicly available information; we did not seek verification with funding bodies. Results: The majority (44/50) of sampled funders indicated funding health research. 38 (of 44, 86%) had publicly available information about disseminating funded research, typically called “policies” (29, 76%). Of these 38, 36 (95%) mentioned journal publication for dissemination of which 13 (36.11%) offer variable guidance on selecting a journal, all of which relate to the funder’s open access mandate. Six funders (17%) outlined publisher requirements or features by which to select a journal. One funder linked to a document providing features of journals to look for (e.g. listed in the Directory of Open Access Journals) and to be wary of (e.g., no journal scope statement, uses direct and unsolicited marketing). Conclusions: Few funders provided guidance on how to select a journal in which to publish funded research. Funders have a duty to ensure that the research they fund is discoverable by others. This research is a benchmark for funder guidance on journal selection prior to the January 2021 implementation of Plan S (a global, funder-led initiative to ensure immediate, open access to funded, published research).", "keywords": [ "journals", "journal selection", "health research funders", "publishing" ], "content": "Introduction\n\nA 2017 investigation found that a considerable number of biomedical research studies supported by well-known funding organizations such as the National Institutes of Health (NIH), were published in so-called “predatory journals”1. Predatory journals are regarded as non-credible and are criticized for failing to provide typical or expected publishing services and their lack of transparent operations2,3. Such services include peer review, long term preservation of content, and indexing in scientific, bibliographic databases. Among predatory journals’ many shortcomings, the failure to ensure permanent, future discoverability of research is a prominent feature threatening the integrity of science. Research that cannot be found by others cannot contribute to science, thus time, money, and resources are wasted1,4. Funders ought to be concerned that funded research may be published in journals that do not ensure discoverability of content ensuring it is available to contribute to future science. Benefits from investments in research are difficult to quantify5. One of the few ways for research funders to measure returns on investments is by tracking research outputs, including scholarly publications6. Predatory journals may limit the scientific return of funders’ investments if their content cannot be reliably found.\n\nMost major funders of health research now mandate that funded research outputs be open access7. Such open access mandates typically require researchers to ensure that research (and sometimes data) is published in an open access journal or is deposited in a publicly accessible digital repository (regardless of whether the publication was published in an open access journal), or both. Some journals, called delayed-access journals, may impose an embargo period only after which an article is made publicly available or can be archived in a repository. Many funders’ policies allow for such delays in open access to accommodate publishers’ preferences.\n\nOpen access policies are one way for funders to direct funded researchers towards publishing in credible journals abiding by established open access tenets8:\n\n1. Research is/should be freely available and accessible to anyone.\n\n2. The copyright accompanying published work should be open, allowing for free use and re-use (i.e., allowing research to be freely built on/adapted with attribution).\n\nTo facilitate researcher adherence with funder open access policies, many biomedical journals offering open access have agreements with the PubMed Central (PMC) repository to automatically deposit their published content on authors’ behalf9. Additionally, researchers funded by the NIH and 13 partner funding organizations in the USA can upload funder-supported publications to PMC from journals without PMC agreements10. Likewise, 29 funders from across Europe can submit funder-supported research to Europe PMC (which is mirrored in PMC)11. For some of these organizations, such as the NIH and Wellcome Trust, archiving in PMC or Europe PMC, respectively, is mandatory.\n\nPredatory journals often market themselves as ‘open access’ and work to aggressively attract submissions12,13. Research in predatory journals may indeed be free to access – if it can be discovered. However, at best, predatory journals’ identification through search engines is sparse and inconsistent, and these journals tend not to be indexed in scientifically-curated, bibliographic databases used by researchers14–16. Rather, predatory journal articles may appear haphazardly in search engines such as PubMed (since it includes author-uploaded articles from PMC)17. Additionally, we do not know whether content in predatory journals will be perpetually available if a journal ceases to operate. Content preservation is typically secured by publishers through service agreements with digital preservation providers (e.g. Lots of Copies Keep Stuff Safe, LOCKSS). For journals indexed in Medline, for example, this is a prerequisite of indexing18; PMC functions as a preservation service (i.e., has a remit to preserve content funded by public money)19. It is unknown whether predatory journals, not formally indexed in Medline, PMC, or other databases with similar requirements, have digital preservation arrangements.\n\nMost researchers have a limited understanding of what open access means beyond making research free to read20–23. Free use and unrestricted re-use of research is a fundamental component of open access and licensing to permit this is a regular component of open access journals24. Journals running nefarious and deceptive publishing operations have likely benefited from or exploited this lack of awareness25. Indeed, few predatory journals mention licensing for articles or provide information on the use and re-use of published research24. Without explicit licensing for published articles, the legalities around distributing or building on research in predatory journals, for example, is uncertain. Whether researchers are deceived by predatory journals or are knowingly seeking easy and rapid publications in them (these journals tend to deliver quicker turnaround time than credible journals due to subpar or non-existent peer review26,27), they are likely breaching the open access policies set out by their funders.\n\nIn January 2017, the Bill & Melinda Gates Foundation implemented a policy mandating open access to research publications and data, without delay for all funded research28. In February 2017, they initiated a one-year partnership pilot with the American Association for the Advancement of Science (AAAS) to enable Gates-funded research to be published as open access in five AAAS journals, including Science29. The Gates-AAAS partnership seemed to inspire several other influential journals (i.e., New England Journal of Medicine, Proceedings of the National Academy of Sciences) to introduce policies ensuring permanent open access for Gates-funded research30.\n\nIn January 2021, a number of international funders (including UK Research and Innovation, the Gates Foundation, Wellcome Trust, and the World Health Organization), led by Science Europe (a group representing funders across Europe), are set to deliver a radical change to the way that funded research is published via Plan S (coalition-s.org). Plan S will, in part, require research funders to mandate open access to funded research through publication in an open access journal or on an open access platform; and require that publications be made available through an open access repository immediately upon publication. To support this, agreed funders will pay the cost of article publishing charges (APCs) (up to a yet unannounced limit) to journals that are immediately and wholly open access (sometimes referred to as ‘gold’ open access).\n\nWhether health research funding bodies, prior to Plan S, provide funded researchers with guidance or support towards selecting publishing journals in line with their policies and which facilitate proper access to research, and whether they monitor such policies, is unknown. Previous studies confirm that many non-commercial health research funders’ have policies requiring open access to completed research or results via publication or otherwise31,32. Yet none seem to have assessed whether funders provide any specific information to researchers to facilitate their choice of publishing journal. For public or charitable funders, providing such guidance or support may be one way of ensuring responsible stewardship of public or donor funds. The current work will establish a pre-Plan S baseline of health research funders’ guidance and recommendations to researchers for selecting journals in which to publish funded research.\n\nThe aim of this research was to describe the policies and recommendations of major health research funding bodies regarding suitable publication venues for funded research.\n\n\nMethods\n\nWe considered the public websites of 50 health funding bodies for statements, guidance, or policies specifically mentioning the publication of funded research. Detailed methods and rationale for this study are elaborated in an a priori study protocol (https://doi.org/10.17605/OSF.IO/J6CSK) and summarized below.\n\nGlobal funding bodies with the largest documented health research expenditures were sampled from the curated Health Research Funder’s list found at: www.healthresearchfunders.org33. The list was developed as part of an unfunded post-doctoral project by researchers in the Netherlands33. It was last updated in 2016; expenditure data are reported in 2013 US dollars (USD, accounting for inflation/deflation rates by country). A detailed account of the systematic process used to identify funders and obtain expenditure data is found here: http://www.healthresearchfunders.org/faq/. At the time of retrieval for this study (August 2017), 287 health research funding bodies from 30 countries were included on the list. The list distinguishes five categories of funders: [1] philanthropic funders (n=194), [2] public funders (n=77), [3] public funders who fund health research through Official Development Assistance (public ODA)1 (n=8), [4] multilateral funders (funding across countries) (n=7), and [5] public-private partnerships (PPP) (n=1) (Table 1). While there are some inequities in its coverage (e.g. public funders were selected from only G20 countries; paucity of funders from low income countries), the list is likely the most comprehensive source of global health research funder expenditure information in existence (personal communication, Dr. Beverley Holmes, Michael Smith Foundation for Health Research) and has been used to construct samples in at least two other studies32,34. This study excludes commercial funders since their expenditure data are not publicly or readily available.\n\nCreated from data at healthresearchfunders.org.\n\n† OECD: Organisation for Economic Co-operation and Development.\n\n‡ Includes 3 funders with no expenditure data available.\n\n§ Includes 11 funders with no expenditure data available.\n\n* Includes 1 funder with no expenditure data available.\n\n†† Includes 5 funders with no expenditure data available.\n\nTo construct our sample, we sought up to 15 funders with the largest expenditures from each of the five funder categories from the list at www.healthresearchfunders.org, and aimed to include all listed lower income countries (n=4) if they were not otherwise represented in the sample. We included the latter group of funders in order to ensure representation from lower income countries, since researchers and journals from these countries have been disproportionately implicated in predatory publishing24,35. Working with the available number of funders in each category (Table 1), we ended up with 50 funders: 15 philanthropic, 15 public, eight public ODA, seven multilateral, one PPP, and four lower-middle income country funders.\n\nIn line with previous investigations into health research funder policies32,36, we expected that guidance for funded researchers would be publicly available and easily obtained. For each included funder, one of us (LS) visited the website using the URL provided by www.healthresearchfunders.org, or if the URL was not working, found it through a Google search using the funder name. When a funder’s website could not be located/did not work or when the funder was a duplicate, the next largest funder on the list was used. For each funder, we sought and downloaded the website section on policies for funded research in August-September 2017. If no specific policies were found, we searched the SHERPA (Securing a Hybrid Environment for Research Preservation and Access)/Juliet database (www.sherpa.ac.uk/juliet/index.php), which lists and links conditions for open access publication for some funders (though this is incomplete as it is reliant on voluntary contributions from funders and other organizations [e.g., libraries] tracking funder policies). If a funder’s website did not mention funding health research (i.e., funded other scientific research) or if the funder did not specifically award grants for research, the funding body was excluded from the sample and replaced with the next largest funder (by expenditure), where possible. Reasons for exclusion are documented in Figure 1.\n\nOne assessor (LS) extracted information from the downloaded policy documents into an online form in Distiller SR, and a second assessor (KDC or MJP) verified the extracted data. Discrepancies were resolved by discussion. If additional documents were identified during extraction, we saved them and searched them for the desired data. The verification process led to clarifications in collected data or provided additional information. The following information was assessed or extracted, as available:\n\nAny statement(s) about the dissemination of outputs from funded research\n\nPolicy or recommendations about publication of funded research\n\nPolicies or recommendations on research/data accessibility\n\nPolicies or recommendations on journal quality, impact factor or other metric, ethical standards, and indexing of funded research;\n\nWhether/what information is provided to researchers about predatory or substandard journals, or about journal credibility\n\nStrength of any aforementioned policies/recommendations (‘must’, ‘should’, or ‘suggested’)\n\nFor publication policies, whether adherence will be monitored\n\nFor publication policies, whether consequences of non-adherence are listed\n\nIf non-English websites or documents were encountered and an English-language version was not available on the website, Google Translate was used to automatically translate the websites and documents. Google Translate has recently shown 85%-97% accuracy across nine languages for translating health research studies37, including the languages encountered in this study (French and German).\n\nWe summarized data descriptively by calculating proportions for dichotomous data; the date of funder policies/recommendations were summarized as medians and interquartile range.\n\nIn the protocol for this study, we stated that we wanted to determine whether there were differences in the number of funders with policies/statements about journal quality and predatory publishing based on the income-level of the funder country or country being funded. However, as only four funders from lower-middle income countries and none from low income countries were on the list we sampled from, there were not enough funders to enable meaningful comparisons between higher income and lower income countries.\n\n\nResults\n\nFor the 50 funding bodies originally identified using the described sampling technique, three allocated money from a funder (European Commission2) already in the sample and were replaced with the next organizations on the list. One of the replacement funders also allocated money from an included funder and was also replaced. Two funders funded non-health research and four funders did not list any research grants (and appeared to fund health development initiatives) and could not be evaluated for our purposes. Overall six funders were excluded and lacked replacements in the categories they belonged to. 44 funders remained in the sample for which grant policies and guidelines were sought (Figure 1). 35 funders are from high income countries, one from upper-middle income (China), three are from a lower-middle income country (India), and five are not classified by income level because they are multilateral (n=3) or fund across the European Union (EU) (n=2, Table 2).\n\ni China.\n\nii All funders from India.\n\niii Income-level not available since funders distributing funds across multiple countries (3 multilateral funders and 2 European Union funders).\n\niv No funders from “Low income” countries in sample.\n\n38 of 44 funders (86%) had publicly available information for grantees about disseminating funded research outputs (Table 3). Of the six funders that did not have publicly available information, five are from high-income countries (US, Germany, France, UK) and one funds research in the EU through public-private partnership. Three are philanthropic organizations and two are public-ODA funders. Information about disseminating research was contained within “policies” for 29/38 (76%) funders, “recommendations” (suggestions and guidance) for 8/38 (21%) of funders, and as a “code of conduct” for one funder (Table 3). All but one policy/recommendation referred to funded research (including results) as the unit of dissemination (37/38, 97%). Over a third of policies/recommendations also specifically mentioned the dissemination of “data” (25/38, 66%). The median implementation date or date listed on collected documents was September 2014 (IQR: Apr 2012 to Apr 2016, n=35).\n\na Denominator = 44 funders with grant guidelines.\n\nb Denominator = 38 funders with statements about research outputs.\n\nc Policy: uses the words “policy”, “must”, “require”; Recommendation/Guideline: uses the words “recommendation” “recommend”, “suggest”, “should”, “guideline”.\n\nd Other - described as “Code of Conduct”.\n\ne Verbatim: activities of funded organizations; all research outputs, news releases; photos; any and all other published material referencing the research project or grant; code; research materials; protocols; research resources including, but are not limited to, the full range of tools that scientists and technicians use in the laboratory, such as cell lines, antibodies, reagents, animal models, growth factors, combinatorial chemistry, DNA libraries, clones and cloning tools (such as PCR), methods, laboratory equipment and machines; presentations; media interviews; and other professional activities; 'research tools'; metadata; bibliographic metadata; supplementary materials; other supporting artefacts, research resources/tools.\n\nf Out of 35 funders listing this information. Date of implementation was used if available, otherwise, date of document or last update was used. When only year was given, January was used as default month; when a date range was given the most recent date was used.\n\n36 of 38 policies/recommendations (95%) specifically referred to publication in a journal as one form of dissemination for completed research (Table 4). 31 of these (86%) mentioned that research should be open access, either through journal publication (n=24, 77%) or through self-archiving the final report or accepted manuscript in a freely accessible repository (such as PMC) (n=30, 97%). One funder from India (Indian Council of Medical Research), one from France (Institut National de la Santé Et de la Recherche Médicale, INSERM), and three from the USA (US Department of Defense, Congressionally Directed Medical Research Program, and the American Cancer Society) did not mention open access in their policies about research dissemination.\n\n1 Denominator: 36 funders mentioning journal publication.\n\n2 See Table 5 for verbatim text of statements about journal selection.\n\n3 See Table 5 for verbatim text of statements about journal credibility.\n\n4 excluding JIF.\n\n5 2 funders indicate journal should be “high quality, peer reviewed journal”; 1 funder indicates journal should be “quality peer-reviewed journal”; 1 funder indicates what a good journal is: \"Good journals have guidelines for reviewers of manuscripts committing them to strict confidentiality/to disclose conflicts of interest and promise to respond to submitted manuscripts within a specified, short time limit, and correspondingly set their reviewers short time limits for their comments.\"\n\n6 See Table 6 for transparency or ethics standards for publications.\n\n7 Other: 1 funder encourages publication in “primary scientific journals”; 1 funder states “models and mechanisms for publication and access to research must be both efficient and cost effective”;.\n\n13 of 36 (36%) policies recommending publication contained some guidance on how to select a journal and six (17%) listed features or requirements of publishers or journals for researchers to look for (Table 5). These six are described here. Only one funder policy (NIH) included a definition of a journal (i.e., either a publication listed in the journal section of the National Library of Medicine or one meeting stated criteria). And only one funder policy (Canadian International Development Research Council, IDRC) appeared to provide any information about ‘questionable’ journals in a guidance document entitled “Publishing in Open Access Journals”. The document lists journal features to “look for” and to “be wary of” and mentions Beall’s List3 as a resource (Table 5). One policy (Deutsche Forschungsgemeinschaft/German Research Foundation, DFG) linked to Think, Check, Submit (www.thinkchecksubmit.org) – an initiative to facilitate researchers’ assessment of the credentials of a journal – on a page supplementing their open access policy listing open access resources. Two funders distributing APC fees through the Charitable Open Access Fund (Cancer Research UK and Wellcome Trust) list the requirements of journals whose APCs are eligible for payment through the fund. The Bill and Melinda Gates Foundation provide researchers with a portal (called Chronos) through which to submit manuscripts directly to pre-selected journals whose standards are in line with their requirements.\n\n* “Open Choice” is a term used by the publisher Springer to refer to hybrid journals.\n\nThe policies of at least three funders (German Federal Ministry of Education and Research Indian [BMBF], Indian Department of Biotechnology [DBT], Indian Department of Science & Technology [DST]) include a statement that further to making research freely accessible, researchers’ choice of journal was unrestricted.\n\nMost funders mentioned that funded research should be peer reviewed or published in a peer reviewed journal (Table 4). Four funders made non-specific reference to selecting a “good” or “quality” journal in relation to publication of funded research; none mentioned the journal impact factor. Eight funders made statements about publication transparency or ethics. For instance, one funder discussed reproducibility in published research, three mentioned adherence to reporting guidelines, and at least six asked that metadata accompany published articles (Table 6).\n\nOf 38 policies/recommendations providing information about disseminating research outputs, only nine (24%) stated that they monitor adherence to either a policy (n=7) or recommendation (n=2); two philanthropic funders (Wellcome Trust and Bill & Melinda Gates Foundation) specified that they would evaluate publications of funded research reported to them to ensure they are published in journals meeting the funder’s outlined publishing requirements (Table 7). No monitoring or adherence data appears to be publicly available. Only five (13%) funders with policies or recommendations about journal publication indicated that there would be consequences for non-adherence. And only two of those (Wellcome Trust and NIH) stated that they would withhold or suspend payments if articles are not made open access.\n\n\nDiscussion\n\nMost health research funders appear to have active policies about the dissemination of funded research, typically in the form of policies about open access which often include statements about journal publication. Few policies provide guidance on how to select a journal, list features of journals meeting funder requirements, or about the credibility of publishing outlets. Only one health research funding organization (IDRC) made specific reference to the “questionable journals” at the time of data collection (August-September 2017). Additionally, few policies describe whether funded outputs are monitored for compliance with funders’ dissemination policies, and few describe any consequences for researchers’ non-adherence to their policies. Information is not available on whether the NIH or Wellcome Trust, both of whom promise to withhold or suspend grant funds for breaching their open access policies, have actually ever done so7.\n\nFor many of the funders in our sample, information to guide publication of research was found across multiple documents and not always within the main open access policy statements/documents. For example, the only funder to provide guidance about predatory journals (IDRC) did so in a PDF (entitled “Publishing in Open Access Journals”) that was linked to from their main open access policy. The policy itself did not flag that the document contained information about predatory/questionable/non-credible journals. This non-obvious placement of guidance or expectations around adherence to their funder policies relies on researchers’ curiosity or knowledge that important information may be located outside of the main policy webpages or documents. If funders wish to provide guidance about journal credibility and predatory publishing, they may reach more researchers (and increase the likelihood of them reading it) by including such information within a single, main policy document aimed at funded researchers.\n\nAt least four previous studies examining health research funder policies on clinical trial transparency have collected information on funder’s recommendations for disseminating research.\n\nTwo studies using similar methods evaluated trial transparency policies (i.e., those related to trial registration, access to summary results, individual data availability) for non-commercial health research funders globally (n=18)32 and in the USA (n=9)34. After accounting for three common funders across studies, 21 of 24 (87.5%) funders (16 of which are represented in our study) either required or supported publication or registration of trial results (neither study or their available data distinguished between publication or registration). This is in line with our findings in which 86% (38 of 44) funders had such policies/recommendations.\n\nA third study, published in 2017 which examined research waste-reducing policies of 11 non-commercial funders (six of which are represented in our study) reported six to be explicit in requiring publication of full reports of funded research31. In comparison, 36 of 38 policies/recommendations (95%) in our study referred to journal publication as one form of dissemination for completed research but did not indicate that it was mandatory. There may be differences in how authors of that study and interpreted language in documents or policies. The names of the six funders ‘requiring’ publication in that study were not obvious in either the publication or available data, so we are unable to investigate this further.\n\nA study published in 2008 examined 73 UK and international non-commercial health research funders’ guidance for reporting funded clinical trials38. 49 funders (67%) explicitly stated that trials could or should be published. Of the three funders appearing in the 2008 sample and ours, all have maintained recommending (but not requiring) the publication of trial results. Whether funders provided any guidance on selecting a journal to publish in was not collected in the study.\n\nNo previous studies appear to have investigated whether health research funders’ provide guidance to help funded researchers select a journal for publication. Our study appears to be the most comprehensive investigation on this matter. This is surprising since our findings suggest that funders in our sample regard publication as the primary means of disseminating funded research. Further, studies show that researchers view journal publication as the primary way of disseminating research39,40.\n\nThis study is the first to examine the information funders provide researchers about selecting a journal in which to publish funded research. All funders in our sample that mention journal publication or provide guidance on selecting journals, do so within their open access policies. In a time where the scholarly publishing landscape has been infiltrated and confused by predatory journals, inadvertently resulting in some researchers trying to achieve open access to publish in predatory journals41, funders can play a critical role in steering researchers in the right direction. Funders can be specific and explicit with regards to which journal features researchers should look for in order to select one that meets their open access requirements.\n\nThis study is an essential benchmark by which to monitor how major health research funders are performing pre and post Plan S implementation (planned for January 2021, at the time of writing). Data collection occurred in August & September 2017, prior to the September 2018 announcement of Plan S. So far, 24 funders have committed to implementing Plan S, five of which were considered in this research (European Commission, Gates Foundation, MRC/UK Research and Innovation, Wellcome Trust, and the World Health Organization). Two of these, the Wellcome Trust and Gates Foundation, provided guidance (in the form of tools) to facilitate selecting a journal in line with their open access policies at the time of sampling. At least one funder (Wellcome Trust) has made changes to their open access policies in anticipation of Plan S42.\n\nOur study relied on publicly available information about funder expectations of funded research and was abstracted by a single person with verification by a second (i.e., not two independent people). Six funders in our sample did not provide any relevant public information. We did not seek verification on policies from funders since data collection took place at a time when the publishing activities, particularly open access, was (and still is) rapidly changing, somewhat in response to the rise of predatory journals41,43. We are aware that the NIH issued a notice on their Public Access Policy in November 2017 (outside of our sampling and data collection period) with recommendations to publish funded research in journals with ‘credible practices’44. The focus of this research is limited to health research funders. We have not accounted for or evaluated other potential scientific publishing gatekeepers such as academic institutions, governments, or companies carrying out scholarly research, despite the important role they can play45.\n\nExplicit funder policies on publication expectations. Selecting a journal in which to publish research is not a straightforward undertaking46, particularly since the emergence of predatory journals. For funders looking to make their expectations around publishing funded research more explicit and more transparent, we propose several recommendations on how this might be achieved in Box 1, based on findings of this research and on the expertise of authors. Providing specific information about journal considerations in funders’ policies to funded researchers may facilitate more thoughtful and responsible selection of journals. Several recommendations in Box 1 pertain to the explicitness of article/journal considerations mentioned in Plan S (e.g., persistent identifiers for publications; long-term preservation/archiving; article-level metadata). All health research funders may wish to consider making aspects of their policies that pertain to publishing more explicit, whether or not they intend to implement Plan S.\n\n\n\n1. Use precise wording to describe your agency’s expectations that funded research be published\n\n- Indicate whether researchers are expected to publish their research (e.g. use of “must” vs “should”)\n\n- indicate whether open access publication is one of several options for meeting the agency’s open access requirements.\n\n2. Provide a definition of a journal that is suitable to your agency\n\n◦ Decide what essential features a publishing entity should and should not have in order to be considered a suitable place for publication.\n\n◦ Consider referring to/including the Committee on Publication Ethics (COPE) list of Core Practices all journals and publishers should follow: https://publicationethics.org/core-practices\n\n◦ The NIH definition of a journal is47:\n\n• Publication meets the requirements for ISSN (International Standard Serial Number) assignment;\n\n• Publication content is issued over time under a common title;\n\n• Publication is a collection of articles by different authors;\n\n• Publication is intended to be published indefinitely.\n\n3. Indicate your agency’s requirements for access and discoverability of published articles\n\n◦ Distinguish between free vs open access:\n\n• Published articles are free to access; AND additionally, for open access,\n\n• Licensing for published articles permit reuse and building on (typically through a Creative Commons Attribution License, CC BY).\n\n◦ Ensure that published research can be accessed in perpetuity\n\n○ Researchers can determine whether the publishing journal has a permanent archival arrangement in place either through automatic deposition to PMC (https://www.ncbi.nlm.nih.gov/pmc/journals/), or to another archive (via the Keepers Registry: https://keepers.issn.org/keepers-registry)xxv\n\n○ PMC-partnered funders can require that researchers upload published research directly to PMC\n\n◦ Journal provides unique permanent identifiers (e.g. digital object identifier [DOI]) (can check if journal/publisher is registered with CrossRef: https://www.crossref.org/06members/51depositor.html)\n\n4. Be clear about your agency’s support for article processing charges arising from publication of funded research\n\n◦ Indicate how much money is available each open access publication (e.g. maximum APC amount)\n\n◦ Indicate who will receive APC payment from the funder – the author (institution) or the journal\n\n◦ Indicate when funding will be distributed to support article processing charges\n\n◦ Indicate whether there are any conditions on distribution of APC funds\n\n5. Indicate whether your agency requires archiving in a repository alongside publication\n\n◦ Indicate whether the publication, data, or both, are expected to be deposited in a repository\n\n◦ Indicate when deposition is expected to occur (i.e., immediately or within a specified time frame)\n\n◦ Indicate whether you have a dedicated repository for research publications (e.g., PMC for NIH-funded research), and if not, suggest one or more repositories that are considered acceptable by your agency\n\n◦ Be clear that it is the authors’ responsibility to ensure publications are deposited in a repository\n\n◦ Provide instructions/link to resources on how to deposit research in the suggested repository.\n\n6. Indicate how your agency will monitor that funded research is published in appropriate journals, in line with agency recommendations/mandates\n\n◦ For ease of monitoring, Provide instructions for researchers about where and how to include the funding agency name and grant number in published articles (guidance here: http://www.rin.ac.uk/system/files/attachments/Acknowledgement-funders-guidance.pdf)\n\n◦ Provide instructions on if, how, and when to submit publications of funded research to the funding agency, or state how publications will be monitored otherwise\n\n◦ Provide specific actions/consequences that the agency will carry out when funded research is published in a journal that does not meet agency requirements\n\nxxvprovides global monitoring of archiving arrangements for electronic journals.\n\nThe NIH is the only funder in our sample to clearly describe what it considers a journal – either those listed in the journal section of the National Library of Medicine (NLM) (https://www.ncbi.nlm.nih.gov/nlmcatalog/journals) or those meeting a comprehensive set of criteria47: (1) meets the requirements for ISSN (International Standard Serial Number) assignment; (2) content is issued over time under a common title; (3) is a collection of articles by different authors; and (4) is intended to be published indefinitely. All but the final criterion are straightforward to judge; presumably it is meant to distinguish a journal from a book or a monograph however NIH or NLM do not provide guidance on how to judge this criterion. However, whether journals intend to publish indefinitely can be predicted or otherwise determined has not been described. A more meaningful criterion for distinguishing journals from non-journals may be whether the publishing entity has archival arrangements in place (e.g., with LOCKSS, Portico, PubMed Central) to ensure perpetual access to content in the event a journal ceases to operate. Since preserving publisher content may have associated costs48, predatory or non-credible journals (which have been described as “primarily fee-collecting operations”49) are potentially unlikely to seek this service.\n\nWe surprised that the three funders from India in our sample (Indian Council for Medical Research, DBT, and DST) did not mention journal credibility or predatory journals, and further, that a common policy for two Indian funders (DST and DBT), dated December 2014, recognizes “the right of researchers to publish their work in journals of their choice, because researchers are the best judges of where to publish their work”. Since at least 2016, there has been an ongoing national initiative combat predatory journals and to support researchers in their choice of journals across higher education institutes in India. The main product of this work has been a list of approved journals in which academic researchers are permitted to publish in as well as standard templates for researchers when communicating with journals50. The University Grants Commission (UGC), the regulator and funder of high education, has been leading the initiative. It is uncertain whether the country’s largest health research funders are on board due to their lack of guidance in this space. A coordinated approach by a range of stakeholder groups45, which includes funders (who have innate authority to implement mandates about publishing), may facilitate improved publication decisions by researchers. Importantly, however, UGC’s list of approved journals has been plagued with numerous credibility concerns in its short existence51,52. Explicit recommendations from India’s funders regarding credible and non-credible features of journals in which to publish may be warranted in the absence of a trusted and comprehensive list.\n\nFacilitating and monitoring adherence to funder policies. Funders are well-positioned to provide researchers with resources and tools to help ensure that results from funded research are published in credible and discoverable journals, in line with their policies. Several organizations in our sample consistently offer more information about potential publishing routes and tools to facilitate adherence to their policies. We provide a list of tools to facilitate the development of funder policies on research outputs, adherence to such policies, and monitoring of policy adherence (Table 8).\n\nxxviPreviously known as FundRef (until Nov 2015).\n\nMonitoring researchers’ adherence to their policies may help funders understand the extent to which researcher’s publishing practices are guided by their policies53. Informing researchers that their adherence to open access policies is being monitored may facilitate better awareness of such policies and potentially increase adherence to them54. A 2018 study examining the accessibility of research supported by 12 research funding agencies across North America and Europe with open access policies, found that 62% of almost 1.3 million articles over nine years were freely available7. In 2016, 90% of published research supported by the NIH and Wellcome Trust was free to access (via journal, repository, or both)7. Both agencies mandate the deposit of published research by publishing journals or funded authors into dedicated repositories (PMC for NIH; PMC Europe for Wellcome Trust). The remaining 10 funders in the sample did not mandate depositing in a repository alongside publication and had lower rates of freely accessible articles. For example, for the Canadian Institutes of Health Research (CIHR) only 55% of published research was freely accessible in 2016, even though the funder had a dedicated repository (PMC Canada) until 2018 (it closed due to low usage and high upkeep costs)55. The study’s authors conclude that funders with low compliance rates used less enforcement and had less infrastructure to support compliance with their open access mandates7.\n\nAn important area of future study is whether researchers are being funded on the basis of grant applications that include research published in predatory journals – or in journals that may not be indexed in trusted databases. Predatory journals have made their way into consideration (via CVs submitted by researchers or through institution-initiated database searches) into applications for academic career advancement56–58. Some have called for such publications to either be discounted from consideration or for researchers who submit them for consideration to be prevented from career advancement overall59,60. It is unknown whether researchers are including publications in predatory journals as part of their funding applications. This should be evaluated. If they are, funders may wish to consider whether this is an important consideration for awarding funding.\n\n\nConclusion\n\nMost large health research funders mandate open access to funded research outputs, typically by way of open access journal publication and by deposition of published research in digital repositories. Few funders provide guidance on what constitutes a journal (or an open access journal) or are checking to ensure that published research that they have funded is indeed meeting specified requirements about how research should be shared. Health research funding organizations have an obligation to support researchers in meeting their mandates so that research can, as intended, contribute to the broader evidence base. The publishing community needs to provide guidance to funders and researchers on universally acceptable and transparent standards for journal operations. Many solutions to improve policies, facilitate adherence, monitor compliance and work with other funders on large-scale improvements exist and should be implemented.\n\nJournals that fail to make research discoverable breach the basic trust that researchers and their funders have in the current publishing system. Most funded researchers publish their work under the basic assumption that their journal or publisher is following best practices to ensure future use61. Bodies funding health research have a responsibility to protect their investments and even more importantly, to ensure that funded research is not wasted by being published in non-credible and non-discoverable sources.\n\n\nData availability\n\nOpen Science Framework: Audit of health research funder policies and recommendations on journal publication of research: Extracted Data, https://doi.org/10.17605/OSF.IO/YUDP462.\n\nThis project contains the following underlying data:\n\n- Funders Data analysis data - clean 2020Apr21.dta\n\nOpen Science Framework: Audit of health research funder policies and recommendations on journal publication of research: Study Forms, https://doi.org/10.17605/OSF.IO/FSUQ263\n\nThis project contains the following extended data:\n\n- Level 1 - Searching funder websites form 2017Nov23.pdf\n\n- Level 2 - Data extraction form 2017Nov23.pdf\n\nOpen Science Framework: Audit of health research funder policies and recommendations on journal publication of research: Protocol, https://doi.org/10.17605/OSF.IO/J6CSK64\n\nRegistration of overarching OSF project: https://doi.org/10.17605/OSF.IO/Z59U665.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nWe would like to thank Dr. Beverley Holmes of the Michael Smith Foundation for Health Research for providing valuable insight into global health research funder operations and policies at the outset of this project.\n\n\nAuthor contributions\n\nThe study was conceived of by LS, JG, and DM. LS drafted the initial protocol, with approval from the study team. LS managed the research project. LS, KDC, and MJP carried out the research. LS cleaned and analysed the data, prepared tables and figures, and drafted the manuscript. All authors provided feedback and gave their approval prior to submission.\n\n\nFootnotes\n\n1ODA is a term coined by the Development Assistance Committee of the Organisation for Economic Co-operation and Development (https://data.oecd.org/oda/net-oda.htm)\n\n2Any European Commission funding program that explicitly stated using the European Horizon 2020 guidelines for grantees were jointly represented as “European Commission”. (Figure 1)\n\n3Beall’s List is an archived listing of potential predatory journals and publishers, as determined by librarian Jeffrey Beall between 2011 and 2017, https://web.archive.org/web/20170103170903/https://scholarlyoa.com/\n\n\nReferences\n\nMoher D, Shamseer L, Cobey KD, et al.: Stop this waste of people, animals and money. Nature. 2017; 549(7670): 23–5. PubMed Abstract \| Publisher Full Text\n\nBeall J: Predatory publishers are corrupting open access. Nature. 2012; 489(7415): 179. PubMed Abstract \| Publisher Full Text\n\nvan Dyck C: The Long-Term Preservation of Open Access Journals. DOAJ News Service. 2018. [cited 2019 Oct 23]. Reference Source\n\nMacleod MR, Michie S, Roberts I, et al.: Biomedical research: increasing value, reducing waste. Lancet. 2014; 383(9912): 101–4. PubMed Abstract \| Publisher Full Text\n\nLane J: Let’s make science metrics more scientific. Nature. 2010; 464(7288): 488–9. PubMed Abstract \| Publisher Full Text\n\nMacilwain C: Science economics: What science is really worth. Nature. 2010; 465(7299): 682–4. PubMed Abstract \| Publisher Full Text\n\nLarivière V, Sugimoto CR: Do authors comply when funders enforce open access to research? Nature. NLM (Medline); 2018; 562(7728): 483–6. PubMed Abstract \| Publisher Full Text\n\nSuber P: Open Access Overview. Focusing on open access to peer-reviewed research articles and their preprints. 2015. [cited 2017 Sep 29]. Reference Source\n\nUS National Institutes of Health: How Papers Get Into PMC. 2015. [cited 2020 Jan 24]. Reference Source\n\nUS National Institutes of Health: PMC and Research Funder Policies. 2019. [cited 2020 Jan 24]. Reference Source\n\nE PMC: Funders - About - Europe PMC. [cited 2020 Jan 24]. Reference Source\n\nMoher D, Srivastava A: You are invited to submit… BMC Med. 2015; 13(1): 180. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nClemons M, de Costa E Silva M, Joy AA, et al.: Predatory Invitations from Journals: More Than Just a Nuisance? Oncologist. 2017; 22(2): 236–40. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNelson N, Huffman J: Predatory Journals in Library Databases: How Much Should We Worry? Ser Libr. 2015; 69(2): 169–92. Publisher Full Text\n\nSomoza-Fernández M, Rodríguez-Gairín JM, Urbano C: Presence of alleged predatory journals in bibliographic databases: Analysis of Beall’s list. El Prof la Inf. 2016; 25(5): 730–737. Publisher Full Text\n\nTopper L: RE: How predatory journals leak into PubMed. CMAJ. 2018. [cited 2020 Jan 8]. Reference Source\n\nManca A, Moher D, Cugusi L, et al.: How predatory journals leak into PubMed. CMAJ. 2018; 190(35): E1042–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUS National Library of Medicine: Fact Sheet MEDLINE® Journal Selection. U.S. National Library of Medicine. [cited 2019 Dec 5]. Reference Source\n\nDOAJ: Applications: a note about Archiving and Preservation. DOAJ Blog. 2015. Reference Source\n\nCarpenter J: Researchers of Tomorrow: The research behaviour of Generation Y doctoral students. Hlava MMK, editor. Information Services & Use. 2012; 32(1–2): 3–17. Publisher Full Text\n\nDallmeier-Tiessen S, Darby R, Goerner B, et al.: Highlights from the SOAP project survey. What Scientists Think about Open Access Publishing. Arxiv. 2011. 1101.5260. Reference Source\n\nCharbonneau DH, McGlone J: Faculty experiences with the National Institutes of Health (NIH) public access policy, compliance issues, and copyright practices. J Med Libr Assoc. 2013; 101(1): 21–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNeylon C: Science publishing: Open access must enable open use. Nature. 2012; 492(7429): 348–9. PubMed Abstract \| Publisher Full Text\n\nShamseer L, Moher D, Maduekwe O, et al.: Potential predatory and legitimate biomedical journals: can you tell the difference? A cross-sectional comparison. BMC Med. 2017; 15(1): 28. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCourt Rules in FTC's Favor Against Predatory Academic Publisher OMICS Group; Imposes $50.1 Million Judgment against Defendants That Made False Claims and Hid Publishing Fees. Federal Trade Commission Press Releases. 2019. [cited 2019 Jun 10]. Reference Source\n\nShen C, Björk BC: ‘Predatory’ open access: a longitudinal study of article volumes and market characteristics. BMC Med. 2015; 13(1): 230. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBjörk BC, Solomon D: The publishing delay in scholarly peer-reviewed journals. J Inf. 2013; 7(4): 914–23. Publisher Full Text\n\nvan Noorden R: Gates Foundation research can’t be published in top journals. Nature. 2017; 541(7637): 270–270. PubMed Abstract \| Publisher Full Text\n\nvan Noorden R: Science journals permit open-access publishing for Gates Foundation scholars. Nature. 2017. Publisher Full Text\n\nvan Noorden R: Science journals end open-access trial with Gates Foundation. Nature. 2018; 559(7714): 311–2. PubMed Abstract \| Publisher Full Text\n\nNasser M, Clarke M, Chalmers I, et al.: What are funders doing to minimise waste in research? Lancet. 2017; 389(10073): 1006–7. PubMed Abstract \| Publisher Full Text\n\ndeVito NJ, French L, Goldacre B: Noncommercial Funders’ Policies on Trial Registration, Access to Summary Results, and Individual Patient Data Availability. JAMA. 2018; 319(16): 1721–1723. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nViergever RF, Hendriks TCC: The 10 largest public and philanthropic funders of health research in the world: what they fund and how they distribute their funds. Health Res Policy Syst. 2016; 14: 12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWhitlock EP, Dunham KM, DiGioia K, et al.: Noncommercial US Funders’ Policies on Trial Registration, Access to Summary Results, and Individual Patient Data Availability. JAMA Netw Open. 2019; 2(1): e187498. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nXia J, Harmon JL, Connolly KG, et al.: Who publishes in “predatory” journals? J Assoc Inf Sci Technol. 2015; 66(7): 1406–17. Publisher Full Text\n\nGoldacre B: The WHO joint statement from funders on trials transparency. BMJ. 2017; 357: j2816. PubMed Abstract \| Publisher Full Text\n\nJackson JL, Kuriyama A, Anton A, et al.: The Accuracy of Google Translate for Abstracting Data From Non-English-Language Trials for Systematic Reviews. Ann Intern Med. 2019; 171(9): 677–679. PubMed Abstract \| Publisher Full Text\n\nDwan K, Gamble C, Williamson PR, et al.: Reporting of clinical trials: a review of research funders’ guidelines. Trials. 2008; 9(1): 66. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAdjei KOK, Owusu-Ansah CM: Publishing preferences among academic researchers: Implications for academic quality and innovation. 2016. Reference Source\n\nWakeling S, Spezi V, Fry J, et al.: Academic communities: The role of journals and open-access mega-journals in scholarly communication. J Doc. 2019; 75(1): 120–39. Publisher Full Text\n\nTopper L, Boehr D: Publishing trends of journals with manuscripts in pubmed central: Changes from 2008-2009 to 2015-2016. J Med Libr Assoc. 2018; 106(4): 445–54. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWellcome updates open access policy to align with cOAlition S \| Wellcome. [cited 2019 Oct 27]. Reference Source\n\nPeterson AT, Johnson PE, Barve N, et al.: The NIH public access policy did not harm biomedical journals. PLoS Biol. 2019; 17(10): e3000352. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNational Institutes of Health (NIH): NOT-OD-18-011: Statement on Article Publication Resulting from NIH Funded Research. [cited 2018 Jan 15]. Reference Source\n\nLalu MM, Shamseer L, Cobey KD, et al.: How stakeholders can respond to the rise of predatory journals. Nat Hum Behav. 2017; 1(12): 852–5. PubMed Abstract \| Publisher Full Text\n\nTenopir C, Dalton E, Fish A, et al.: What Motivates Authors of Scholarly Articles? The Importance of Journal Attributes and Potential Audience on Publication Choice. Publications. 2016; 4(3): 22. Publisher Full Text\n\nNational Institutes of Health: Plan for Increasing Access to Scientific Publications and Digital Scientific Data from NIH Funded Scientific Research. 2015. Reference Source\n\nMorris S: The true costs of scholarly journal publishing. Learn Publ. 2005; 18(2): 115–26. Publisher Full Text\n\nBerger M, Cisarella J: Beyond Beall’s List: Better understanding predatory publishers. Coll Res Libr News. 2015; 76(3): 132–5. Publisher Full Text\n\nTao T: India’s Fight Against Predatory Journals: An Interview with Professor Bhushan Patwardhan. The Scholarly Kitchen. 2020. [cited 2020 May 1]. Reference Source\n\nPatwardhan BK, Nagarkar S, Gadre SR, et al.: A critical analysis of the ‘UGC-approved list of journals’. Curr Sci. 2018; 114(6): 1299–303. Publisher Full Text\n\nPatwardhan B: Why India is striking back against predatory journals. Nature. 2019; 571(7763): 7. PubMed Abstract \| Publisher Full Text\n\nGraham ID, Logan J, Harrison MB, et al.: Lost in knowledge translation: time for a map? J Contin Educ Health Prof. 2006; 26(1): 13–24. PubMed Abstract \| Publisher Full Text\n\nMichie S, Richardson M, Johnston M, et al.: The Behavior Change Technique Taxonomy (v1) of 93 Hierarchically Clustered Techniques: Building an International Consensus for the Reporting of Behavior Change Interventions. Ann Behav Med. 2013; 46(1): 81–95. PubMed Abstract \| Publisher Full Text\n\nAnderson K: Lessons from the Demise of PubMed Central Canada. Caldera Publishing Solutions. 2018. Reference Source\n\nSeethapathy GS, Santhosh JU, Hareesha AS: India’s scientific publication in predatory journals: need for regulating quality of Indian science and education. Curr Sci. 2016; 111(11): 1759–1764. Publisher Full Text\n\nKolata G: Many Academics Are Eager to Publish in Worthless Journals. The New York Times. 2017. Reference Source\n\nBagues M, Sylos-Labini M, Zinovyeva N: A walk on the wild side: ‘Predatory’ journals and information asymmetries in scientific evaluations. Res Policy. 2019; 48(2): 462–77. Publisher Full Text\n\nCappell MS: List predatory journal publications separately from genuine scholarly publications as standard for CVs. BMJ. 2015; 350: h2470. PubMed Abstract \| Publisher Full Text\n\nPond BB, Brown SD, Stewart DW, et al.: Faculty applicants’ attempt to inflate CVs using predatory journals. Am J Pharm Educ. American Association of Colleges of Pharmacy; 2019; 83(1): 7210. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDe Solla Price DJ: Little science, big science. Columbia Univ. Press; 1963; 119. Publisher Full Text\n\nShamseer L, Brehaut J, Stewart LA, et al.: Extracted Data. 2020. http://www.doi.org/10.17605/OSF.IO/YUDP4\n\nShamseer L, Cobey K, Page MJ, et al.: Study Forms. 2021. http://www.doi.org/10.17605/OSF.IO/FSUQ2\n\nShamseer L, Cobey K, Page MJ, et al.: Study Protocol. 2021. http://www.doi.org/10.17605/OSF.IO/J6CSK\n\nShamseer L, Cobey K, Page MJ, et al.: Audit of health research funder policies and recommendations on journal publication of research. 2021. http://www.doi.org/10.17605/OSF.IO/Z59U6" }	[ { "id": "79367", "date": "01 Mar 2021", "name": "Shubhada Nagarkar", "expertise": [ "Reviewer Expertise Bibliometrics", "publication ethics", "institutional repositories." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe purpose of this work is clear and the work is explicitly and precisely presented. Researchers would like to describe the policies of major health research funding agencies. The job is sound technically.\nAuthors selected policies from 44 funding agencies and assessed systematically to find information and recommendations on the dissemination of research output, recommendations on criteria for the selection of journals (quality, impact factor and other metrics).\nMethodology for data collection and analysis is discussed very well and the protocol followed is described in detail.\nStatistical data can be interpreted easily. Source data can be reproducible.\nThe results obtained through this exercise are well discussed and will be of interest to all funding agencies, especially those from developing countries. The findings of the study show that there is no specific guidance about how to select a journal for publication. The mention and cautionary notes on predatory publishers and their open-access nature should be made clear by these funding agencies. Authors warned policymakers about this. If the policy of these funding agencies advises researchers to current trends in publishing, researchers may not fall into pray with predatory publishers.\nOverall, this work is recommended for indexing. Similar studies can be considered for the assessment of policies of funding agencies other than health research funders.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6522", "date": "16 Apr 2021", "name": "Larissa Shamseer", "role": "Author Response", "response": "Thank you for your thoughtful comments." } ] }, { "id": "79363", "date": "08 Mar 2021", "name": "Nicholas Devito", "expertise": [ "Reviewer Expertise Metascience", "health policy", "publication ethics." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMany thanks for the opportunity to review this piece on health research funders’ policies on journal selection for research outputs.\nOverall, this piece investigates an interesting topic, has appropriate methods, appears well-executed, and follows open science best principles of pre-registration and open sharing of data for which it should be commended. I ultimately believe it should be recommended for indexing but a minor revision is in order. Specific points are provided below.\nCan the authors please revisit the piece for a bit of editing? The informational content all appears very relevant and well-researched but the prose can carry on at times and structurally it jumps back and forth between topics and it is difficult to connect the threads. This is most applicable to the introduction (and perhaps the Discussion as well) but I think taking a critical eye towards developing a more direct, succinct, and straight-forward writing style throughout would enhance readability. A sharper consolidation and organisation of your ideas and arguments, while trying to be more concise overall, will aid readers considerably. A brief example: “Funders ought to be concerned that funded research may be published in journals that do not ensure discoverability of content ensuring it is available to contribute to future science” could surely be consolidated to something like “Funders should ensure published research is discoverable to the scientific community.”\n\nOne area that doesn’t really come through clearly in the Intro is what funders get out of publication of their funded work and how this influences this dynamic. Why are they moving towards Open Access? What about other dissemination routes?\nThe personal communication citation of Dr. Beverly Holmes feels a bit out of place. Is she merely stating an opinion? If so, why can the authors not just state this opinion directly themselves since I assume they share it? I’m sure Dr. Holmes is perfectly knowledgeable and her opinion is valuable, but is citing her necessary here? I’m most familiar with citations of personal communication to convey official information not published elsewhere, from a direct source, rather than simply used to state an opinion the authors agree with. The average reader won’t have any insight as to why they should trust the word of Dr. Holmes on this issue.\nThe link to the protocol is upfront and clear and points readers to exactly where they can easily access more detailed information about the search/data extraction methods. Very well done by the authors.\nHow were the extraction criteria derived? Did the authors base them on anything or did they come up with them de novo? This should be stated and potentially explored in the Discussion.\nOne major area I was surprised that the authors did not include in their assessments is whether the funder offers to pay the open access/APCs of work they fund. This would be an important part of the dynamic of how, where, and why certain journals may be chosen and why funders should care even more about where the research they fund ends up.\nIn Figure 1, it is unclear when INSERM is mentioned as to whether that is the funder being removed or the reason another funder is being removed (later context shows it’s the latter, but this is unclear). Also there is a type (“allocated”) in the “Excluded Funders n=6” box. I think ideally you would list which exact funders are entering or exiting the sample in this figure for clarity as you don’t get a good sense of what funders are actually being assessed until the results. It’s also not necessary to restate in prose everything Figure 1 already conveys (something to be cognisant of throughout).\nTable 5 is a bit overwhelming. Perhaps consider summarising each policy in Table 5 and make the full text extractions available as an appendix for interested readers.\nI don’t find the reason given for a lack of outreach to sponsors particularly compelling. The author’s state they did not verify with sponsors (despite this being a feature of the work from Goldacre et al. they cite as a template for this research) because “data collection took place at a time when the publishing activities...was rapidly changing.” That seems like a post hoc justification rather than an actual reason why it didn’t actually occur. It also isn’t supported by their own data as the median date of effect of policies reported was September 2014, 3 years prior to data collection and the higher IQR range is April 2016, over a year before the searches. Similarly, this article is cross-sectional, so I don’t see that reason as relevant anyway. You could simply have discarded policies from before a cutoff date, etc. Can the authors please revisit their discussion of this limitation?\nTable 8 may fit better in an appendix.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6523", "date": "16 Apr 2021", "name": "Larissa Shamseer", "role": "Author Response", "response": "Thank you for reviewing this work. Please see responses to your comments below. We have made some changes in the introduction and discussion sections to make the wording more concise. We have clarified that scholarly publishing is intended to facilitate the discoverability and uptake of funded research (into practice) and that predatory journals limit this by having unreliable discovery and questionable trustworthiness. We did not set out to investigate funders’ open access (OA) policies or shifts towards OA since discoverability and other journal features compromised by predatory journals can be achieved independent of openness. We note that several sources (ROARMAP, SHERPA JULIET) list details of funders' open access policies. This research is instead concerned with hallmarks of journal validity which have been (mis)taken as signals of research quality. Regarding other dissemination routes, we have clarified that this research focuses on journal publications rather than other publications routes (end of introduction section). As indicated, Dr. Holmes is the CEO of a health research funding organization (not included in our sample). Her expertise of the funding landscape and whether a listing of health research funders existed or whether the chosen list was the best source of this information, was an invaluable methodological contribution to our research. We prefer not to remove this acknowledgment. Extraction criteria were devised de novo based on expertise within the author team. There is no reference standard for funder policies about publishing and as such we did not use any framework for extraction. We have added a clarification of this point in the methods section rather than in the discussion section. We have added text to the introduction (2nd para) outlining the waste of research funds & APCs due to predatory journals. While we did not extract specific data on funders' APC provisions, where funders provided information about journal APC support, this can be found in Table 5. We have updated the suggested information in the flow diagram and left the related descriptive text in results for additional reference. We did not apply an analytical framework to the extracted text and doing so would be a post-hoc analysis. The table/text is provided for reference/transparency of extracted data summarized in Tables 2-4. We do not feel that a summary is necessary. To be clear, we did not make a post-hoc justification regarding the lack of outreach to funders. We specifically did not have the a priori intent of verifying our data with funders. We anticipated that the results of our previous work, https://www.nature.com/news/stop-this-waste-of-people-animals-and-money-1.22554, which identified a large number of funders supporting research in predatory journals, may impact/prompt changes in funder policies once published (Sept 2017). Due to the timing of the current study, (Aug – Sept 2017), we decided not to engage funders since we did not want to influence any potential changes to their policies as a result of the study process. We did not find this to be a limitation. As expected, and as noted in our discussion, we are aware of at least one funder (NIH) who, in Nov 2017, clarified their policies about publication as a result of the previous study. The specific impacts (i.e., number and type of clarifications) of engaging funders to clarify extracted data in Goldacre et al’s previous work are unclear, however, we have added text to the limitations section outlining the additional potential benefit of engaging funders in the study process. F1000 does not allow appendices and we have opted not to move this table to the Open Science Framework repository for this project as supplementary materials. We feel that the recommendations in this table are part of our discussion and are likely to receive little uptake if readers have to navigate away from the full text." } ] } ]	1	https://f1000research.com/articles/10-100
https://f1000research.com/articles/10-289/v1	15 Apr 21	{ "type": "Research Article", "title": "Improved chromosome-level genome assembly and annotation of the seagrass, Zostera marina (eelgrass)", "authors": [ "Xiao Ma", "Jeanine L. Olsen", "Thorsten B.H. Reusch", "Gabriele Procaccini", "Dave Kudrna", "Melissa Williams", "Jane Grimwood", "Shanmugam Rajasekar", "Jerry Jenkins", "Jeremy Schmutz", "Yves Van de Peer", "Xiao Ma", "Jeanine L. Olsen", "Thorsten B.H. Reusch", "Gabriele Procaccini", "Dave Kudrna", "Melissa Williams", "Jane Grimwood", "Shanmugam Rajasekar", "Jerry Jenkins", "Jeremy Schmutz" ], "abstract": "Background: Seagrasses (Alismatales) are the only fully marine angiosperms. Zostera marina (eelgrass) plays a crucial role in the functioning of coastal marine ecosystems and global carbon sequestration. It is the most widely studied seagrass and has become a marine model system for exploring adaptation under rapid climate change. The original draft genome (v.1.0) of the seagrass Z. marina (L.) was based on a combination of Illumina mate-pair libraries and fosmid-ends. A total of 25.55 Gb of Illumina and 0.14 Gb of Sanger sequence was obtained representing 47.7× genomic coverage. The assembly resulted in ~2000 unordered scaffolds (L50 of 486 Kb), a final genome assembly size of 203MB, 20,450 protein coding genes and 63% TE content. Here, we present an upgraded chromosome-scale genome assembly and compare v.1.0 and the new v.3.1, reconfirming previous results from Olsen et al. (2016), as well as pointing out new findings.\n\nMethods: The same high molecular weight DNA used in the original sequencing of the Finnish clone was used. A high-quality reference genome was assembled with the MECAT assembly pipeline combining PacBio long-read sequencing and Hi-C scaffolding. Results: In total, 75.97 Gb PacBio data was produced. The final assembly comprises six pseudo-chromosomes and 304 unanchored scaffolds with a total length of 260.5Mb and an N50 of 34.6 MB, showing high contiguity and few gaps (~0.5%). 21,483 protein-encoding genes are annotated in this assembly, of which 20,665 (96.2%) obtained at least one functional assignment based on similarity to known proteins. Conclusions: As an important marine angiosperm, the improved Z. marina genome assembly will further assist evolutionary, ecological, and comparative genomics at the chromosome level. The new genome assembly will further our understanding into the structural and physiological adaptations from land to marine life.", "keywords": [ "Seagrass", "Zostera marina", "eelgrass", "chromosome-scale genome assembly", "annotation" ], "content": "Introduction\n\nSeagrasses are a polyphyletic assemblage of early-diverging monocotyledoneous plants belonging to the Alismatales (Les, Cleland, and Waycott 1997; Du and Wang 2016); they are not true grasses (Poaceae). Several clades of seagrasses arose independently from freshwater sister taxa 3-4 times between the Paleocene and late Eocene (~65-34 mya) and are the only fully marine, flowering plants (~14 genera and ~65 species) (Chase et al. 2016). They occur in predominantly soft-sediment, marine coastal environments worldwide (Green, Short, and Frederick 2003) and as engineering species provide the foundation of three-dimensional habitats that are among the most productive and biodiverse (Costanza et al. 1997; Duffy et al. 2015). Seagrass meadows provide numerous ecosystem services, e.g., provisioning of fish and invertebrates, retention of nutrients (Larkum, Orth, and Duarte 2006) and carbon sequestration (Fourqurean et al. 2012). Unfortunately, they are also under threat related to human impacts (Waycott et al. 2009) that fundamentally change coastal system dynamics (Duffy et al. 2015) and make restoration extremely difficult (van Katwijk et al. 2016). Effective marine conservation strategies require integrative research perspectives between ecology and genomics (Hillebrand, Jacob, and Leslie 2020) because ecological and evolutionary change can and do occur on the same time scales (Carroll et al. 2007), e.g., genetic polymorphisms underlying critical traits or the role of genetic diversity at selectively relevant sites for population resilience.\n\nZostera marina (eelgrass) is a marine model species with >3000 papers covering a wide variety of ecological, evolutionary, conservation and biotech topics. Its unique, circumglobal, warm temperate to Arctic distribution has allowed it to withstand numerous cycles of rapid climate change during the Pliocene glacial and interglacial periods (Olsen et al. 2004), empirically demonstrating its capacity to adapt, acclimate and recover (Duarte et al. 2018), e.g., to temperature (Franssen et al. 2011; Jueterbock et al. 2016; Jueterbock et al. 2020), salinity gradients/osmoregulation (Shivaraj et al. 2017), ocean acidification (Zimmerman 2020) and potential pathogens (Brakel et al. 2014; Guan, Saha, and Weinberger 2020; Zang et al. 2020). Further, clonal populations of Z. marina can persist for hundreds to thousands of years ((Thorsten et al. 1999) for Z. marina, (Arnaud-Haond et al. 2012) for Posidonia oceanica)), yet have found ways to adapt through periods of rapid climate selection via genotypic plasticity, fostered by somatic mutation (Yu et al. 2020) and epigenetic modification of the methylome (DuBois, Williams, and Stachowicz 2020; Jueterbock et al. 2020). Microbiome interaction studies are being conducted in parallel with eelgrass resequencing, e.g., bacterial (Cucio et al. 2016; Fahimipour et al. 2017; Wang, Tomas, and Mueller 2020; Eisen et al. 2017) and fungal (Ettinger, Voerman, et al. 2017; Ettinger, Williams, et al. 2017; Ettinger and Eisen 2019, 2020; Ettinger, Vann, and Eisen 2020) to inform restoration strategies as well as meta-organismal function. Bioengineered salinity tolerance is also of interest (Wani et al. 2020).\n\nThe new assembly of the Z. marina reference genome will further advance studies in the aforementioned areas, as well as comparative analyses of genome structure and evolution, as new reference genomes for representatives of the other three seagrass lineages (i.e., Posidonia oceanica - Posidoniaceace, Cymodocea nodosa - Cymodoceaceae and Thalassia testudinum - Hydrocharitaceae) come online in the near future along with Zostera mueller (Lee et al. 2016) and Zostera japonica (unpublished).\n\n\nMethods\n\nWe used an aliquot of the same DNA that served as the basis for Z. marina v.1.0 genome. We sequenced the Z. marina genome using a whole genome shotgun sequencing strategy and standard sequencing protocols. Sequencing reads were collected using Illumina and PacBio platforms at the HudsonAlpha Institute for Biotechnology in Huntsville, Alabama, USA. Illumina reads were sequenced using the Illumina HiSeq-2500 platform and the PacBio reads were sequenced using the SEQUEL II platform. One 400 bp insert 2×150 Illumina fragment library (162.7× coverage), and one 2×150 Hi-C Illumina library were constructed using Dovetail Hi-C kit and sequenced to 581.1× coverage. Prior to assembly, Illumina fragment reads were screened for PhiX contamination. Reads composed of >95% simple sequence were removed. Illumina reads <50 bp after trimming for adapter and quality (q<20) were removed. The final read set consists of 280,181,449 reads for a total of 156.4× of high-quality Illumina bases. For the PacBio sequencing, a standard PacBio long read library was constructed and a total of 8 PB chemistry 3.0 chips (10 hours movie time) were sequenced on a Sequel 1, a sequence yield of 75.97 Gb, with a total coverage of 189.93x.\n\nThe current v.3.1 assembly was generated by assembling the 5,615,408 PacBio reads (189.93x sequence coverage) using the MECAT assembler (Xiao et al. 2017) and subsequently polished using ARROW (Chin et al. 2013).\n\nMisjoins in the assembly were identified using Hi-C data as part of the JUICER pipeline (Durand et al. 2016). No misjoins were identified in the polished assembly. The contigs were then oriented, ordered, and joined together using HI-C data using the JUICER pipeline. A total of 89 joins were applied to the assembled contigs to form the final set of six chromosomes. Each chromosome join is padded with 10,000 Ns. Significant telomeric sequence was identified using the (TTAGGG)n repeat, and care was taken to make sure that it was properly oriented in the production assembly. The remaining scaffolds were screened against bacterial proteins, organelle sequences, GenBank nr and removed if found to be a contaminant.\n\nFinally, homozygous SNPs and INDELs were corrected in the released consensus sequence using 40x of Illumina reads (2x150, 400bp insert) by aligning the reads using bwa mem (Li 2013) and identifying homozygous SNPs and INDELs with the GATK’s UnifiedGenotyper tool (McKenna et al. 2010). A total of 1,876 homozygous SNPs and 64,447 homozygous INDELs were corrected in the release.\n\nRepeatModeler v2.0 was used to build a custom repeat library for the genome assembly of Z. marina v.3.1. Subsequently, RepeatMasker v4.1 was used to discover and classify repeats based on the custom repeat libraries from RepeatModeler v2.0. Transfer RNAs (tRNA) were predicted by tRNAscan-SE v1.31 (Chan and Lowe 2019) with default parameters. We also identified noncoding RNAs, such as microRNAs (miRNAs), small nuclear RNAs (snRNAs) and ribosomal RNAs (rRNAs) by comparing with known noncoding RNA libraries (Rfam v14.2), using the cmscan program of Infernal v1.1.2 (Nawrocki and Eddy 2013). In addition, novel miRNA entries from the Z. marina v.1.0 assembly were aligned to hard-masking Z. marina v.3.1 using SeqMap (Jiang and Wong 2008) with no mismatches. We extracted ~ 110 bp upstream and downstream sequences surrounding every aligned locus and discarded the miRNAs candidates located within protein coding sequences or repetitive elements (“NNNNNNNNNNN”). The stem-loop structure and the minimum free energy (MFE) were predicted for each region using the RNAfold program of the ViennaRNA v 2.1.1 (Lorenz et al. 2011) with default settings. Finally, the results based on Rfam and Z.marina v.1.0 were combined into a non-redundant prediction of miRNAs.\n\nGenome annotation was performed using a combination of ab initio prediction, homology searches and RNA-aided alignment. Augustus (Stanke, Tzvetkova, and Morgenstern 2006) was used for ab initio gene prediction using model training based on protein structures and RNA-seq data from Z. marina v.1.0 (Olsen et al. 2016). For homology-based predictions, the protein sequences of Z. marina v.1.0 and Oryza sativa were downloaded from PLAZA (https://bioinformatics.psb.ugent.be/plaza/) and aligned to Z. marina v.3.1 using TBLASTN with different e-values (Z. marina v.1.0 with e-value ≤ 1e-10 and O. sativa with e-value ≤ 1e-5). Next, regions were mapped by these query sequences to define gene models using Exonerate (Slater and Birney 2005). For RNA-aided annotation, we downloaded 23 libraries of Z. marina v.1.0 from NCBI (BioProject PRJNA280336). Firstly, we joined the paired-end reads using clc_assembly_cell to generate almost 2/3 of joined reads and 1/3 of un-joined reads. Then, we aligned the joined and un-joined RNA-seq data to Z. marina v.3.1 using HISAT2 (Kim, Langmead, and Salzberg 2015) with the parameters “--max-intronlen 50000” and assembled into potential transcripts using StringTie v2.1.1 (Kovaka et al. 2019). Further, TransDecoder v5.0.2 was used to predict open reading frames (ORFs) within the assembled transcripts. Finally, gene models obtained from all three approaches were integrated as the final non-redundant gene set using EVidenceModeler (v.1.1.1) (Haas et al. 2008). Specifically, a set of 1,460 bad gene models identified by the wgd software (Zwaenepoel and Van de Peer 2019) was manually curated using the genome browser GenomeView on the ORCAE platform (https://bioinformatics.psb.ugent.be/orcae/) and the gene annotation results were evaluated by BUSCO hits. Putative gene function was determined using InterProScan (Jones et al. 2014) with the different databases, including PFAM, Gene3D, PANTHER, CDD, SUPERFAMILY, ProSite, and GO. Meanwhile, functional annotation of predicted genes was also obtained by aligning the protein sequences against the sequences in public protein databases (Uniprot/SwissProt database) using BLASTP with an e-value cut-off of 1×10-5.\n\n\nResults and discussion\n\nA single genotype (or clone) of Z. marina from the northern Baltic Sea, Finnish Archipelago Sea had been subjected to whole-genome assembly using Sanger and Illumina sequencing (referred to as Z. marina v.1.0) (Olsen et al. 2016). Since PacBio technology can deliver longer reads, necessary to improve assembly contiguity and obtain a nearly complete, reference genome, we re-sequenced the inbred, Finnish clone, leading to the final v.3.1 release, which contains 260.5 Mb of sequence, consists of 432 contigs with a contig N50 of 7.0 Mb and a total of 87.6% of assembled bases into six pseudo-chromosomes (2n = 12). Interestingly, during the assembly of the genome using Hi-C, it was noted that there was a seventh “chromosome” (scaffold_7 in this release) with a length of 8.68Mb, consisting of mainly repetitive DNA and a possible Nucleolus Organizing Region (NOR). Completeness of the euchromatic portion of v.3.1 assembly was assessed using 20,450 annotated genes from Z. marina v.1.0. The screened alignments indicate that 20,342 (99.47%) of the previously annotated version 1.0 genes aligned to the v.3.1 release. Of the remaining genes, 50 (0.24%) aligned at <50% coverage, while 58 (0.29%) were not found in the v.1.0 release. This shows a high degree of consistency between the two versions. However, version 3.1 presents much higher contiguity and fewer gaps compared to the previously published Z. marina v.1.0 (Table 1).\n\nWe used ab initio approaches to identify and annotate repetitive sequences, which accounted for 67.12 % of Z. marina v.3.1. 41.72 % of these TEs were long terminal repeat (LTR) elements (Table 2). Screening the Z. marina v.3.1 genome against the Rfam v14.2 database using Infernal identified 546 tRNAs, 376 rRNAs, 93 miRNAs, and 134 snRNAs (Table 3). In addition to the 93 known conserved miRNAs identified from Rfam v14.2, we also identified 23 novel miRNAs candidates compared to 19 novel miRNAs candidates in Z. marina v.1.0, resulting in a total of 116 miRNAs (Table 4).\n\nThrough a combination of ab initio prediction, homology searches and RNA-aided evidence, we annotated 21,533 gene models after masking repeat elements. After manually checking most gene models and improving 1395 incorrect gene models on the ORCAE platform and adding 30 new genes based on RNA-seq evidence, the final annotation produced 21,483 gene models, 91.8% of which (19,739 genes) are supported by transcriptome data from leaves, roots and flowers. On average, protein-coding genes in Z. marina v.3.1 are 3,300 bp long and contain 4.99 exons, values that are very similar to those of Z. marina v.1.0. Notably, intron lengths greatly improved after manual curation (Table 5). BUSCO assessment of the current gene set shows that the current annotation includes 95.7% complete genes in the embryophyte database10. 93.2% of the BUSCO genes were single copy while 2.5% of these BUSCO genes were found in duplicate. 0.5% of the BUSCO genes were fragmented and 3.8% was missing, which could be due to some specific pathways missing in Z. marina, compared to land plants (Olsen et al., 2016) (Table 6). BUSCO assessment in Z. marina v.3.1 shows more complete genes and fewer fragmented genes than for Z. marina v.1.0 (Figure 1). 20,665 genes (96.2%) obtained at least one functional assignment based on similarity to known proteins in the databases. Pfam domain information could be added to 15,716 (73.2%) predicted genes, and 12,406 (57.7%) predicted genes could be assigned a GO term (Table 7).\n\n\nConclusions\n\nHere, we report a high-quality, chromosome-scale genome assembly of Z. marina using a combination of single-molecule real-time sequencing and Hi-C scaffolding. Although a draft genome sequence for Z. marina has been available for more than five years (Olsen et al. 2016), a chromosome-scale assembly and well-annotated reference genome is an important step to further advance our understanding with respect to its metabolism, evolution and adaptation. As we expected, there is a discrepancy in genome size between an Illumina-derived assembly and a PACBIO long read-derived assembly, which is mainly due to the more accurate coverage of repetitive content. Nevertheless, the genome size of 259 Mb still characterizes Z. marina as relatively compact monocot genome, and it is also the smallest genome among the seagrasses where genome size estimates exist (JGI pilot analyses). Also, telomere and centromere regions are generally captured more fully. As a first high quality, reference genome of an important marine angiosperm, v.3.1 of the genome of Z. marina will provide a great resource for further comparative and evolutionary studies.\n\n\nData availability\n\nNCBI BioProject: Zostera marina Genome sequencing and assembly. Accession number: PRJNA701932; https://identifiers.org/ncbi/bioproject:PRJNA701932.\n\nThe genome assembly and annotation for Z. marina v.3.1 is also available from https://data.jgi.doe.gov/refine-download/phytozome?organism=Zmarina and through the ORCAE platform (https://bioinformatics.psb.ugent.be/gdb/zostera/).", "appendix": "Acknowledgements\n\nWe thank Shengqiang Shu from the Joint Genome Institute for validating the annotation.\n\n\nReferences\n\nArnaud-Haond S, Duarte CM, Diaz-Almela E, et al.: Implications of extreme life span in clonal organisms: millenary clones in meadows of the threatened seagrass Posidonia oceanica. PLoS One. 2012; 7: e30454. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "83351", "date": "16 Jun 2021", "name": "Jarkko Salojärvi", "expertise": [ "Reviewer Expertise Genomics", "population genetics", "bioinformatics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present a new assembly of eelgrass based on long read sequencing and Hi-C chromosome conformation capture technology. Altogether this is a well-prepared improvement on the existing genome assembly. In addition to improving the contiguity, also the manual curation of 1,460 gene models is very much welcome.\nFor comparison purposes the authors could still produce a dot plot of the whole genome alignments of the old vs new version.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "89904", "date": "29 Sep 2021", "name": "Vratislav Peska", "expertise": [ "Reviewer Expertise Telomere biology", "genomics", "sequencing" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript \"Improved chromosome-level genome assembly and annotation of the seagrass, Zostera marina (eelgrass)\" by Ma, Olsen, Reusch et al. is a well-written article about a unique genome of marine angiosperm plant Z. marina. The results convincingly present improving the genome assembly using PacBio and Hi-C strategies. The improvement is obvious, among others, from an increased number of coding genes by 1000 in comparison with the first assembly and almost removing fragmented hits in the BUSCO analysis results.\nI must say that I do not have any serious concerns about the methodology, results, and conclusions. The release of an improved genome is a great opportunity of the research community to have a powerful tool in genome-dependent studies of this extraordinary plant.\nMy minor notes:\nIn the text, \"come online in the near future along with Zostera mueller\" probably should be \"muelleri\".\n\nThe sentence, \"The remaining scaffolds were screened against bacterial proteins, organelle sequences, GenBank nr and removed if found to be a contaminant.\" would need rewording to be clear what \"GenBank nr\" means. Does it mean anything that is not obviously of Zostera species origin?\n\nI suggest using the \"BWA-MEM algorithm\" instead of \"bwa mem\".\n\nI do not understand what the (“NNNNNNNNNNN”) stands for.\n\nI think a short explanation of the assembly numbering would be helpful for readers. Was the Zosma2.0 published and is available somewhere? The JGI link shows v2.2 and v3.1. Actually v3.1 contains data named Zmarina_668_v2.0.fa.gz. The ugent link shows only V2. In summary, the numbering is confusing and a bit more intuitive naming would be nice.\n\nIn Peska et al., (2020)1, we showed there are two telomerase RNA genes in Z. marina. I wonder if assembly v3.1 confirms them as a set of separate genes or two alleles of the same gene? We also showed decompacted mitotic chromosome at the rDNA locus which might be a fragile site. Does it correspond to the observed \"extra\" pseudochromosome in the v3.1?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-289
https://f1000research.com/articles/10-287/v1	13 Apr 21	{ "type": "Systematic Review", "title": "Other ways of communicating the pandemic - memes and stickers against COVID-19: a systematic review", "authors": [ "Jeel Moya-Salazar", "Betsy Cañari", "Lucía Gomez-Saenz", "Hans Contreras-Pulache", "Betsy Cañari", "Lucía Gomez-Saenz" ], "abstract": "Background: In the midst of the coronavirus disease 2019 (COVID-19) pandemic, there are many ways to communicate hygiene measures, such as memes and stickers that are widely used on social networks. We carried out a systematic review in order to determine the impact of stickers and memes as tools to face the COVID-19 pandemic, following the PRISMA guide. Methods: The search was carried out in scientific databases (MEDLINE / PubMed, ScientiDirect, Scielo, LILACS, and Latindex), and in public pre-publication servers (bioRxiv, SocArXiv, medRxiv and Preprints). The publications were identified using the terms (((meme) OR (sticker)) AND ((COVID-19) OR (SARS-COV-2)) AND (WhatsApp)) and the corresponding translations for Spanish and Portuguese. Results: In the initial search, 8434 studies were obtained, 7749 in Preprints, 446 in SocArXiv, 145 in ScientDirect, 82 in medRxiv, and 12 in PubMed. No studies were found in LILACS, Latindex, Scielo, or bioRxiv. Of the 51 studies included as eligible, all were eliminated for not meeting the study inclusion criteria. The majority (40 studies) were eliminated as studies were publications related to the social aspects related to COVID-19, but did not develop an analysis of stickers or memes. Conclusions: No studies were identified that met the inclusion criteria related to the role of stickers and memes as tools to face the COVID-19 pandemic. More studies are needed to estimate its role as a means of communication in health.", "keywords": [ "Smartphone", "COVID-19", "eHealth Strategies", "Cell Phone Use", "SARS-CoV-2", "Health Promotion" ], "content": "Introduction\n\nIn the entire history of humanity, around 20 pandemics have been posed a danger to global health and have shaken the health systems of each era. The recent coronavirus disease 2019 (COVID-19) pandemic, caused by the new type of coronavirus (SARS-CoV-2), is causing high rates of infections and mortality in almost all countries in the world.1 The difference between COVID-19 which is ~12 months-old, and its equivalents such as the Spanish flu (1918-1919), the Black Death (1347-1321), or smallpox (1520), is that COVID-19 develops in the middle of the technological era where natives and digital immigrants coexist in an environment of fundamental interconnection.\n\nIn the midst of a videocracy, governments worldwide have developed primary and secondary prevention mechanisms against this pandemic. The main tools in almost all affected countries, in the absence of vaccines or standardized treatments, have been quarantine (social isolation) and the use of optimal hygiene measures (through hand washing and social distancing).2 To understand both prevention strategies, it is necessary for communities to integrate these “new” concepts as informational and educational tools, and apply them to their daily lives.\n\nThe media undertake this purpose by providing massive amounts of information about COVID-19, monopolizing all broadcast schedules. However, language as a primary tool for this purpose should not only be represented by the use of textual-written language. The brain, being a semiotic system, has the ability to read various forms of language (mathematical formulas, icons, cinema, figures, etc.), and in fact it does, more so now that people are influenced by the stimuli that the image offers. It is understood that we read more than language that only depends on letters and words.3 For this reason, communications increasingly hover over “new” forms of language such as the use of symbols and figures (emojis). Its benefits and challenges have even been considered in medical communications.4\n\nApparently, these images are not limited to the definitions established by language, and on the contrary they generate a more expressive and cosmopolitan language.5 Like emojis, stickers also seem to represent a key piece of contemporary communication, including in providing information on prevention measures (such as hand washing) during the COVID-19 pandemic (Figure 1).\n\nAs epidemic multimodal characters,6 stickers have been considered by the World Health Organization (WHO) as an important tool to face the pandemic, detailing basic prevention measures and changes in social behavior, such as new citizen behaviors of distancing (Figure 2). In collaboration with WhatsApp, the WHO developed a set of stickers in April 2020 called “Together at home” in 10 languages, seeking to highlight the work of health professionals, prevention measures (such as hand washing), and activities promoted during confinement.\n\nAlthough stickers have a growing impact as tools for the dissemination of prevention measures against COVID-19, memes have not yet been considered as tools for the same purpose, even though memes are massively used one-person productions that reflect the current moment with ironic interpretations.\n\nIn this study, we conducted a systematic review to determine the impact of stickers and memes as tools to face the COVID-19 pandemic.\n\n[Has been reproduced respecting the intellectual property rights of WhatsApp sticker users].\n\n[Has been reproduced respecting the intellectual property rights of the World Health Organization’ WhatsApp stickers].\n\n\nMethods\n\nIn order to assess the role of stickers and memes in the face of the current pandemic, we developed a systematic review following the recommendations of the PRISMA guide (Preferred Reporting Items for Systematic Reviews and Meta-Analyzes).7,23 We consider eligible publications that met the following inclusion criteria: (i) original studies (prospective or retrospective), trials, narrative reviews, case-control studies, perspectives, and scientific letters, and (ii) articles in English, Portuguese, and Spanish. We consider as exclusion criteria articles of reflection, systematic reviews, meta-analysis, and editorial letters (correspondence). This systematic review was registered in PROSPERO (CRD42020210205, date: 12/16/2020), complying with international requirements in good research practices and publications of systematic reviews.\n\nWe carried out a systematic search in the main scientific search engines (PubMed, Scopus, Scielo, ScientDirect, LILACS, and Latindex), in public pre-publication servers (bioRxiv, SocArXiv, medRxiv and Preprints) and in Google Scholar and Yahoo! Pre-publication search engines were used since the global emergency context led to the promptness of publication with the extensive use of these search engines.\n\nThe publications were identified using the terms (((meme) OR (sticker)) AND ((COVID-19) OR (SARS-COV-2)) AND (WhatsApp)) and the corresponding translations for Spanish and Portuguese [(((meme) OR (sticker) OR (stickers)) AND ((COVID-19) OR (SARS-COV-2)) AND (WhatsApp))]. The manual search was carried out between December 22 and 30, 2020 and the review was carried out independently by two authors to verify the inclusion criteria of the studies according to PRISMA (Figure 3).\n\nWe consider as standard studies those that detailed the impact of memes and/or stickers on the promotion and prevention of COVID-19, those that described the types of memes and stickers used in social networks, and the organizations that promote their use.\n\nData extraction was performed by two investigators (J.M-S and H.C-P.) according to the predefined protocol. The studies were included in a data matrix (in IBM SPSS v21.0, Armonk, USA) according to the objectives of the study, this base was checked twice by the researchers. A formal descriptive analysis of the studies found was carried out.\n\nPRISMA flow chart for the selection of studies on stickers and memes against COVID-19.\n\n\nResults\n\nIn the study selection, the initial search obtained 8454 studies, consisting of 8277 in Preprints, 446 in SocArXiv, 145 in ScientDirect, 82 in medRxiv, 20 in Scopus and 12 in PubMed. No studies were found in LILACS, Latindex, Scielo, or bioRxiv. Of the 51 studies included as eligible, all were eliminated for not meeting the study inclusion criteria. The majority (40 studies) were eliminated as studies were publications related to the social aspects related to COVID-19, but did not develop an analysis of stickers or memes. Other publications included a newspaper account about quarantine8 and the use of WhatsApp in times of pandemic (but without relevance for the use of memes and stickers).9,10\n\nDuring the study period, no studies were identified that meet the standard required to analyze the impact of stickers and memes as communication tools to face the COVID-19 pandemic, therefore there is insufficient firm evidence that can explain their impact on programs of prevention, given the null evidence found, despite the fact that many governments and international institutions are using these communication elements as information strategies in the face of the current pandemic.\n\n\nDiscussion\n\nThis systematic review showed that there is no evidence available on the role of stickers and memes as tools to face the COVID-19, despite the fact that many governments and international institutions are using these message elements as communication strategies against the current pandemic.\n\nBy not obtaining eligible studies, this study is classified as an “empty review”.11 The empty reviews are also tools to improve existing evidence. Like scoping reviews,12 these reviews are particularly important since they show the null existence of studies on the area of interest (the role of memes and stickers against COVID-19). We well know that current health strategies are using all the available technological resources to face the inclemency of the pandemic, however, there are few reports on the role of digital media actors today.13 The limitation of this study was that thesis or in-house institutional publications were not included since we focus on the searching the main databases.\n\nSince digital communication began several decades ago, mechanisms have been established to provide rapid, massive, secure, and free access to communication using the benefits of the internet. With the explosion in popularity of digital telephones and the growth of applications (apps), the world is becoming interconnected within the imperative need for human communication.\n\nWhatsApp (Facebook, MA, US) allows free instant mobile messaging through texts, phone calls, videos, and also allows sharing multimedia files individually, or in both national and international groups.5 More than a billion people currently use this application in many fields, including in the university setting.14,15 This system has an easy-to-use platform that shares the same mobile contacts, allows communication in different formats (text, voice messages, images, emojis, and recently stickers and moving stickers (gif)) clearly dominating instant messaging worldwide.\n\nStickers (artistic adhesives), which were initially introduced in digital communication by Line (LINE corp., Tokyo, Japan) with payments for access to these, and then Telegram (Telegram FZ-LLC, London, UK) with a more open sticker platform, so that the user can design their own stickers and can share them. This influenced WhatsApp’s adoption of stickers, since 2018, as a language for all its users, whether they are millennials, centenials, etc., enabling creativity in their design that surpasses the Unicode of emojis.\n\nStickers as a powerful communication tool (where users can create, modify and share at their convenience) and the number of WhatsApp users provide an inclusive and massive communication environment of the personal and the social.16 The stickers are not limited to the definitions that language establishes, and on the contrary they generate a more expressive and cosmopolitan language. Stickers have a gradually relevant role in digital communication, replacing other forms of language.17\n\nJust as emojis4,18 can offer new advantages in health communication, stickers seem to represent a key piece of contemporary communication, even in providing information on hand washing during the current COVID-19 pandemic. That is why WhatsApp has been used to share hygiene messages and prevention measures in quick and interchangeable language. Figure 1 shows the free access stickers created by users (in March 2020) with the desire to promote the prevention of COVID-19 in Spanish. These stickers, and many others in other languages, undoubtedly show a persuasive form of free access communication related to the current context of the global health emergency.\n\nThis was quickly supplemented by the WHO, which is also using stickers on WhatsApp to promote healthy behaviors (Figure 2). By considering stickers exclusively in digital communication via WhatsApp, the WHO highlights the popularity of this non-verbal communication format and uses the full benefits of technology in favor of public health. The fact that an international entity can promote authorized and accurate information on the COVID-19 pandemic, not only allows education and promotes hygiene during the pandemic, but could also face the infodemic that affects the understanding of the current disease by civil society.19 We believe that stickers are, in that sense, tools against COVID-19 and “fake news”.\n\nOn the other hand, memes are also potentially vehicles to understand the visceral manifestations of populations, as they are created by people in the exercise of their digital citizenship, they record a historical time (in which they occur) in a particular cultural way. Thus, memes are elements of a chronological record of understanding the world, in short, as an intrinsically literary element. Then, the meme can give us an account of the story of a phenomenon.20,21\n\nAs the findings of this study demonstrate, nothing has yet been described about the meaning of reading a meme in health emergency contexts. We recently explained the differences between what a meme means (or what it can mean) and what it means to read a meme (what happens from the neural network to subjective mental experience when a person reads a meme) (J Moya-Salazar 2021, personal communication, 04 March). Memes become unique in communicative terms because when they are read, an exponential, inadvertent, enveloping and personal laugh comes to them.22\n\nThe phenomenon of memes in the COVID-19 pandemic can also constitute a personal channel, which health institutions can use to promote health promotion activities, correct information, dissemination of hygiene measures, and disease prevention, as previously shown in other digital environments.13\n\n\nConclusions\n\nTo the date of the systematic review (December 30, 2020), no studies were identified that met the inclusion criteria related to the role of stickers and memes as tools to face the COVID-19 pandemic. These new forms of language could be important tools to inform about COVID-19 prevention strategies, since social networks via the internet are massive, fast, and interconnect population groups in real time.\n\nThe prevention strategies that governments (and civil society) are currently using are based on these “new” forms of language to undertake strategies and meet prevention indicators.\n\nThis review indicates that there are no recent studies on the subject, therefore, to estimate the role and impact of these new forms of communication in health it is necessary to develop future studies in order to improve the available scientific evidence.\n\n\nData availability\n\nNo data are associated with this article.\n\nFigshare: PRISMA checklist for ‘Other ways of communicating the pandemic: memes and stickers against COVID-19’. https://doi.org/10.6084/m9.figshare.14204123.v1.23\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nYi Y, Lagniton PNP, Ye S, et al.: COVID-19: what has been learned and to be learned about the novel coronavirus disease. Int J Biol Sci. 2020; 16(10): 1753–1766. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWorld Health Organization: WHO guidelines on hand hygiene in health care. Geneva: WHO; 2009.\n\nOrtiz P: Lenguaje y habla personal. El cerebro humano como sistema semiótico. In:1ed. Fondo Editorial Universidad Nacional Mayor de San Marcos: Lima; 2002.\n\nO’Reilly-Shah VN, Lynde GC, Jabaley CS: Is it time to start using the emoji in biomedical literature? BMJ. 2018; 363: k5033. Publisher Full Text\n\nSutikno T, Handayani L, Stiawan D, et al.: WhatsApp, Viber and Telegram: which is the Best for Instant Messaging? Int J Elect Comp Eng. 2016; 6(3): 909–914. Publisher Full Text\n\nCarmelino AC, Kogawa L: Stickers do Whatsapp: (nova) forma persuasiva de interação bem-humorada. Rev Eletrôn Est Integ Disc Argumentação. 2020; 20(1). Publisher Full Text\n\nMoher D, Liberati A, Tetzlaff J, et al.: The PRISMA Group Preferred Reporting Items for Systematic Reviews and Meta-Analyses: The PRISMA Statement. PLoS Med. 2009; 6(7): e1000097. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMunyikwa M: My COVID-19 diary. Antropol Today. 2020; 36(3): 16–19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGebbia V, Piazza D, Valerio MR, et al.: Patients with Cancer and COVID-19: A WhatsApp Messenger-Based Survey of Patients' Queries, Needs, Fears, and Actions Taken. JCO Glob. Oncol. 2020; 6: 722–729. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDuong TA, Velter C, Rybojad M, et al.: Did Whatsapp® reveal a new cutaneous COVID-19 manifestation? J Eur Acad Dermatol Venereol. 2020; 34(8): e348–e350. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYaffe J, Montgomery P, Hopewell S, et al.: Empty Reviews: A Description and Consideration of Cochrane Systematic Reviews with No Included Studies. PLoS One. 2012; 7(5): e36626. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMunn Z, Peters MDJ, Stern C, et al.: Systematic review or scoping review? Guidance for authors when choosing between a systematic or scoping review approach. BMC Medical Res Methodol. 2018; 18(1): 143. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBecerra-Chauca N, Taype-Rondan A: TikTok: A new educational tool in the fight against COVID-19? Acta Med Peru. 2020; 37(2): 249–251. Publisher Full Text\n\nMartínez MM: La transmisión del lenguaje no verbal en WhatsApp. [Thesis] España. Universidad de Jaén: Facultad de Humanidades y Ciencias de la Educación; 2019.\n\nMaffett PG: El fenómeno Whatsapp en espacios universitarios (Universidad Pública de El Alto, 2019). Edu Sup. 2020; 7(1): 46–61.\n\nAl-Maroof RS, Salloum SA, Hamadand MA, et al.: A Unified Model for the Use and Acceptance of Stickers in Social Media Messaging. In: Hassanien A, Shaalan K, Tolba M (Eds). Proceedings of the International Conference on Advanced Intelligent Systems and Informatics 2019. AISI 2019. Advances in Intelligent Systems and Computing; 2020; 1058.\n\nCarmelino AC, Kogawa L: Stickers do Whatsapp: (nova) forma persuasiva de interação bem-humorada. Rev Eletrôn Est Integ Disc Argumentação. 2020; 20(1). Publisher Full Text\n\nLotfinejad N, Assadi R, Aelami MH, et al.: Emojis in public health and how they might be used for hand hygiene and infection prevention and control. Ant Res Infect Cont. 2020; 9(1): 27. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZaracostas J: How to fight an infodemic. Lancet. 2020; 395(10225): P676. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWells DD: You all made dank memes: using internet memes to promote critical thinking. J Polit Sci Educ. 2018; 14(2): 240–248. Publisher Full Text\n\nBañuelos CJ, Pérez RB: Memes e imaginarios sociales mexicanos en Copa del Mundial de la FIFA 2018. MHCJ. 2020; 11(1): 97–115. Publisher Full Text\n\nRodden A: Neuroscience: What makes us laugh. Nature .2011; 473(7348): 450. Publisher Full Text\n\nMoya-Salazar J: PRISMA checklist. figshare. Dataset. 2021. Publisher Full Text" }	[ { "id": "84657", "date": "20 May 2021", "name": "Kai Zhang", "expertise": [ "Reviewer Expertise Single-cell genomics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an “empty review”. The authors conducted a systematic review to determine the impact of stickers and memes as tools to cope with the COVID-19 pandemic. In order to find relevant studies, they performed a systematic search in six scientific search engines, four pre-print servers, and two general search engines. Among 8454 publications found by the automated search, none of them were considered relevant to this review study according to their inclusion criterion. Their inclusion criterion is reasonable. The fact that this review is “empty” may reflect that this area of study is very new and has not been sufficiently studied, highlighting a potential research gap in this field.\nI have no specific comments for the authors.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [] }, { "id": "83266", "date": "15 Jun 2021", "name": "Jacob I. Bañuelos Capistrán", "expertise": [ "Reviewer Expertise Image", "communication", "technology and culture." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article explores whether there are studies related to alternative ways of communicating content related to the pandemic caused by COVID-19, such as memes or stickers, through an analysis of scientific publications related to the subject. The study finds that there are no studies on these forms of communication associated with the COVID-19 pandemic. The find is interesting. It is recommended to delve further into a detailed description and typology of these alternative forms and ignored by scientific studies on communication.\nThe study has methodological rigor and provides interesting data on studies related to the use of stickers and memes to disseminate distancing and hygiene measures during the COVID-19 pandemic.\nCorrection: The word \"Artículos\" must be translated into English in Fig. 3.\nIt is suggested to include a translation of the legends that accompany the stickers in Fig. 1.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-287
https://f1000research.com/articles/10-136/v1	22 Feb 21	{ "type": "Research Article", "title": "Knowledge and compliance with Covid-19 infection prevention and control measures among health workers in regional referral hospitals in northern Uganda: a cross-sectional online survey", "authors": [ "Sharon Bright Amanya", "Richard Nyeko", "Bonniface Obura", "Joy Acen", "Caroline Nabasirye", "Rebecca Nakaziba", "Florence Oyella", "Victor Afayo", "Mark Okwir", "Richard Nyeko", "Bonniface Obura", "Joy Acen", "Caroline Nabasirye", "Rebecca Nakaziba", "Florence Oyella", "Victor Afayo", "Mark Okwir" ], "abstract": "Background: Infection prevention and control (IPC) has increasingly been underscored as a key tool for limiting the transmission of coronavirus disease 2019 (Covid-19) and safeguarding health workers from infections during their work. Knowledge and compliance with IPC measures is therefore essential in protecting health workers. However, this has not been established among health workers in northern Uganda in light of the Covid-19 pandemic. The objective of this study was to determine the knowledge and compliance with Covid-19 infection prevention and control measures among health workers in regional referral hospitals in northern Uganda. Methods: An online cross-sectional descriptive study was conducted among 75 health workers in regional referral hospitals within northern Uganda. A structured questionnaire was distributed to health workers via WhatsApp messenger. Sufficient knowledge was considered at a correct response score of ≥80%, while adequate compliance was rated ≥75% of the maximum score. Data were analyzed using SPSS v21.\n\nResults: The majority of the health workers had sufficient knowledge (69%) and adequate compliance (68%) with Covid-19 IPC. Adequate compliance was significantly associated with training in Covid-19 IPC (p=0.039), access to Covid-19 IPC at workstations (p=0.036), and having strong institutional support (p=0.031). However, there was no significant relationship between knowledge and compliance with IPC (p=0.07). The socio-demographic characteristics of health workers, including age, sex, education level, occupation, working hours and work experience, had no statistically significant relationship with Covid-19 IPC knowledge or compliance. Conclusion: Our results show fairly good knowledge and compliance with Covid-19 IPC among health workers in northern Uganda. There is need for more training and provision of guidelines to promote compliance with Covid-19 IPC.", "keywords": [ "Infection prevention and control", "knowledge", "training", "compliance" ], "content": "Introduction\n\nCoronavirus disease 2019 (Covid-19) is arguably the greatest global health threat of our time. As of January 24, 2021, 19,462,112 people were infected globally, with 2,112,759 deaths1. In Uganda, the cases are on the rise, with 39,044 cases as of January 24, 20211. As the case numbers grow, health workers are increasingly at risk of infection as they care for the ever -growing number of Covid-19 patients. Because the safety of health workers is key to winning the fight against the virus, infection prevention and control (IPC) measures remain critical tools.\n\nThe World Health Organization (WHO) issued interim guidance on Covid-19 IPC that emphasized several measures, including applying standard precautions to all patients, ensuring early triage and case recognition, and applying additional precautions such as wearing masks2. As the Covid-19 pandemic grows, countries have further stepped up IPC measures, including mandatory wearing of face masks and hand washing in all public places3,4. Without compliance, however, these measures will not help in achieving the intended goal, and the health workers will increasingly be at risk of Covid-19 infection, a fact becoming evident in Uganda where health workers have been infected5.\n\nUganda has designated treatment sites for Covid-19 patients at regional referral hospitals6 where rigorous IPC standards are implemented. However, some asymptomatic Covid-19 patients are likely to seek care from non-designated hospital departments where IPC measures might be inadequate. We believe that health workers in these departments are much more at risk of Covid-19 as they could be managing undiagnosed Covid-19 patients and therefore may not feel compelled to practice strict Covid-19 IPC measures. In this study, we evaluated the knowledge and compliance with Covid-19 IPC measures among health workers in regional referral hospitals in northern Uganda.\n\n\nMethods\n\nA descriptive cross-sectional online study was conducted among health workers at regional referral hospitals (RRHs) in northern Uganda. The RRHs include: Lira RRH, Arua RRH, and Gulu RRH, which serve as referral centers for the 30 districts in northern Uganda. The health workers who participated in the study were those assigned to hospital departments other than the treatment center. Data collection took place between July and August 2020.\n\nThe study targeted health workers whose work involves primary contact with patients, including doctors, nurses, midwives, clinical officers, and laboratory officers. All health workers present at the health facility within the data collection period were included in the study. We exclude health workers who were on study leave and those who were working in designated Covid-19 treatment centers during the data collection period.\n\nThe sample size was determined using the single population proportion formula7 based on assumed probability of good knowledge regarding Covid-19 IPC of 69%8, with a marginal error of 5%, and a standard normal value corresponding to 95% certainty, and a non-response rate of 15%. This yielded a sample size of 213 health workers. Of the 213 health workers approached, only 75 responded within the 1 month period for data collection giving a response rate of 35%.\n\nConvenience sampling was used to identify and select WhatsApp groups with the health workers of interest from the respective RRHs. The researchers obtained verbal consent from the group administrators and requested their help in mobilizing members on the WhatsApp platforms. A link to the questionnaire on Google forms (Alphabet Inc., California, USA) was shared with the potential respondents via WhatsApp messenger (Facebook, Inc., California, USA). Participants were invited to voluntarily participate in the study by following the link shared.\n\nData was collected from 75 health workers using an anonymous, self-administered, online, structured questionnaire adapted from the literature. Knowledge and institutional support were assessed using an eight-item questionnaire adapted from Haridi et al.9 and modified to assess Covid-19 IPC knowledge among health care workers. Compliance was assessed using an eight-item questionnaire from the WHO protocol for the assessment of potential risk factors for Covid-19 infection among health care workers10. The data collection tool consisted of four sections. The first section captured the demographic characteristics of the participants. The second section comprised eight questions ascertaining the level of knowledge and understanding of the concepts of Covid-19 IPC and was scored as follows: one (1) point was awarded for each correct response and zero (0) for an incorrect response, and a correct response score of ≥80% was considered sufficient knowledge. The third section comprised eight questions to ascertain the level of compliance with IPC measures and scored as follows: 1, for ‘never’; 2, for ‘rarely’; 3, for ‘sometimes’; and 4 for ‘always’, giving a possible score of 32 points. Adequate compliance was set at ≥75% (24) of the maximum score. The fourth section comprised three questions concerned with the perception of institutional commitment to IPC and was rated on a Likert scale (never, rarely, sometimes, and always). A scoring system was assigned as follows: 1, for ‘never’; 2, for ‘rarely’; 3, for ‘sometimes’; and 4 for ‘always’, giving a total score of 12. Strong institutional support was considered with a score of ≥75% (9).\n\nEthical approval for this study was obtained from St. Mary’s Hospital Lacor Research and Ethics Committee [Ref. No. LHIREC 0168/06/2020]. The study participants were availed with adequate information regarding the study purpose and procedures and thereafter invited to sign an online informed consent.\n\nThe responses from Google forms were downloaded in an Excel sheet (Microsoft Inc. Albuquerque, New Mexico, United States) and then exported to Statistical Package for the Social Sciences (SPSS) software, version 21.0 (SPSS, Chicago, IL, USA) for analysis. Frequencies and percentages were used to summarize knowledge and compliance with IPC among health care workers, while means and standard deviations were used to summarize data on age, work experience, and other numerical variables. The chi-square test was performed to determine the relationship between categorical variables, while the Pearson correlation test was used to determine the association between continuous variables. A variable was considered significant in this analysis if it had a p-value <0.05.\n\n\nResults\n\nA total of 75 health care workers responded, and the majority were female (52%). More than half of the respondents (60%) belonged to the age group of 20–39 years with a mean age of 36.92 (SD ±9.39). The majority of the health workers interviewed had a bachelor’s degree as their highest level of education and had a mean work experience of 10.4 years (SD ± 8.79). Of all the participants, only 50.7% reported to have received training in Covid-19 IPC, and 66.7% reported having Covid-19 IPC guidelines at their workstations. A significant number of respondents (94.7%) perceived themselves as being at risk of Covid-19 (Table 1).\n\nCovid-19, coronavirus disease 2019; IPC, infection prevention and control; OPD, outpatient department; SD, standard deviation.\n\nThe majority of the respondents (69.3%) had sufficient knowledge, with a mean knowledge score of 5.88/8 (SD ±1.05) (Table 2). Knowledge varied by item assessed. The vast majority of health care workers provided correct responses to items concerning the cleaning of frequently touched surfaces (97.3%), use of contact precautions (94.7%), screening for Covid-19 signs and symptoms (94.3%), applicability of standard precautions (92%), and cleaning of shared equipment (89%). Few correct responses were obtained for items regarding airborne precautions (41.3%) and applicability of hand hygiene (57.4%) (Underlying data file 1). There was no statistically significant relationship between knowledge and socio-demographic variables of respondents (Table 3).\n\n*Statistically significant.\n\nCI, confidence interval; Covid-19, coronavirus disease 2019; IPC, infection prevention and control; SD, standard deviation.\n\nMajority of the respondents (68%) had adequate compliance, with a mean score of 27.35/32 (SD±3.3) (Table 2) and varied by item assessed (Underlying data file 2). Compliance was associated with having received training in Covid-19 IPC (p=0.039), having Covid-19 IPC guidelines at workstations (p=0.036), and sufficient institutional support (p=0.031). There was no statistically significant relationship seen between compliance and sociodemographic characteristics of participants such as age, level of education, working hours, work experience and health worker cadre (Table 3).\n\nGenerally, there was strong perceived institutional support, with the majority of participants (70.7%) feeling adequately supported by their respective institutions. Moreover, strong institutional support was associated with Covid-19 IPC compliance (p=0.031) (Table 3). Just as knowledge and compliance, institutional support score varied by item assessed (Underlying data file 3). For example, only 18.7% of the participants reported always being availed with adequate personal protective equipment (PPE) by their hospitals, while 50.5% reported always having access to handwashing facilities and products, and 49.3% reported always being availed sufficient supplies for the collection of medical waste (Underlying data file 3).\n\n\nDiscussion/Conclusion\n\nIn this study, we evaluated COVID-19 infection prevention control knowledge and compliance among health workers at RRHs in northern Uganda. Our findings suggest that the majority of the health workers in RRHs in northern Uganda are knowledgeable and compliant with Covid-19 IPC. We have identified that 69.3% of the respondents have sufficient knowledge of Covid-19 IPC and that it varied by item assessed. Whereas limited data exist on knowledge of Covid-19 IPC among health workers in Africa, our findings are comparable to those of a similar study at a Chinese mental institution that showed a knowledge score of 63.9%11. However, pre-Covid-19 pandemic studies have shown lower knowledge scores12,13. Taken together, these findings imply that training in IPC during the Covid-19 pandemic has enhanced health workers’ IPC knowledge and therefore needs to be sustained. Moreover, similar to previous studies9,14,15, we demonstrate that knowledge on various parameters of IPC varies. In our study, we found that most health workers were less knowledgeable about parameters related to airborne precautions and hand hygiene. This limited knowledge suggests a need to focus on training, audit and feedback methods to improve knowledge in these areas, as they are central Covid-19 IPC. Furthermore, we did not find a statistically significant relationship between knowledge and participants’ sociodemographic characteristics, IPC training or presence of guidelines. However, previous studies have reported a positive association between knowledge of IPC and age, years of experience, training in IPC, and availability of guidelines14,16. One possible reason for this discrepancy is the context in which the studies were conducted. Unlike these previous studies, our study was conducted during the Covid-19 pandemic, where health workers have been exposed to various training and information sources regarding IPC in the setting of Covid-19.\n\nWe also found that the majority of the health workers (68%) had good compliance with Covid-19 IPC measures. The high score of self-reported compliance is comparable to a previous self-report study14. However, previous studies that used observation methods for data collection reported lower compliance rates; for example, a hand hygiene compliance score of 53–57%11,12,16. This discrepancy could be due to the difference in methods, as self-reported studies are likely to find more compliance with IPC. Moreover, compliance scores also varied by item assessed, as reported in previous studies17,18. In addition, compliance was found to be associated with having had training in Covid-19 IPC, having IPC guidelines, and perceived strong institutional support. Previous studies have shown that training in IPC and access to guidelines improves compliance with IPC12,13,16. Accordingly, the WHO and the Uganda Ministry of Health have emphasized training of all health care staff and developed and supplied guidelines for IPC4,19. These efforts are likely to have contributed to the high knowledge and compliance scores noted in our study. Therefore, our findings provide support for the notion that support to health care workers in terms of training, provision of guidelines and appropriate facilities and supplies for IPC increases compliance.\n\nIn the present study, we also report strong institutional support (70%) for health workers. Despite the high scores, fewer health workers reported adequate provision of PPE. Indeed, inadequate supply of PPE has been a key challenge in health care systems worldwide during this pandemic, with policy makers advocating for the provision of more PPE to protect health care workers20,21. In this regard, Uganda is not spared as the Covid-19 cases and hospital admissions continue to grow. As of January 24th, there were more than 39,000 cases of Covid-19 reported in Uganda1. More so, over 1200 health workers had been infected with the disease as of November 24th, 202022. Our findings point to the need for an adequate and consistent supply of PPE to RRHs in northern Uganda. Nonetheless, the small sample size coupled with the self-report method of measuring compliance are key limitations of this study, and we therefore suggest that further studies consider observation methods to improve the objectivity of the data.\n\nIn conclusion, there is generally good knowledge and compliance with Covid-19 IPC among health workers in regional referral hospitals within northern Uganda. Majority of the respondents (69.3%) had sufficient knowledge on Covid-19 IPC while 68% had adequate self-reported compliance with IPC. Moreover, compliance was associated with health workers having had training in IPC and having Covid-19 guidelines available at their workstations as well as institutional support. These findings suggest a need for more training on a regular basis as well as up-to-date guidelines to ensure compliance with Covid-19 IPC. This limits the spread of Covid-19 to health workers their patients.\n\n\nData availability\n\nOpen Science Framework: [Knowledge and compliance with Covid-19 infection prevention and control measures among health workers in regional referral hospitals in northern Uganda], DOI: 10.17605/OSF.IO/84KJG\n\nThis project contains the following underlying data:\n\n- Underlying data file 1 (Knowledge score by item assessed to determine the knowledge of Covid-19 infection prevention and control among health workers at regional referral hospitals in Northern Uganda)\n\n- Underlying data file 2 (Compliance score by item assessed to determine health workers’ compliance with Covid-19 infection prevention and control measures)\n\n- Underlying data file 3 (Institutional Support for Covid-19 infection prevention and control score by item assessed)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n[Open Science Framework]: [Knowledge and compliance with Covid-19 infection prevention and control measures among health workers in regional referral hospitals in northern Uganda] [DOI: 10.17605/OSF.IO/84KJG]\n\nThis project contains the following extended data:\n\n- Questionnaire (questionnaire used to collect data in this study)\n\nStudy data file (SPSS data file with the raw data collected from participants)\n\nThe authors confirm that they have received permission to reproduce the above questionnaire from the owner of the original questionnaire.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nWe acknowledge the hospital administrators who enabled access to the target population as well as the health care workers for sparing time to participate in the study.\n\n\nReferences\n\nWorld Health Organization: COVID-19 Weekly Epidemiological Update. World Health Organization; 2021. Reference Source\n\nWorld Health Organization: Infection prevention and control during health care when novel coronavirus (‎‎‎nCoV)‎ infection is suspected: interim guidance, January 2020. World Health Organization; 2020. Reference Source\n\nMuseveni YK: Updates on matters regarding Coronavirus (Covid-19) ON 18th May 2020. In: House S, editor. Nakasero. 2020.\n\nMinistry of Health Uganda: UGANDA MINISTRY OF HEALTH COVID-19 INFECTION PREVENTION AND CONTROL GUIDANCE FOR HIV SERVICES DELIVERY. 2020. Reference Source\n\nWHO African Region: Covid-19 Situateion Update for the WHO Africa Region, External Situation Report-23. World Health Organization; 2020. Reference Source\n\nMinistry of Health: National guidelines for Managment of Covid-19 in Uganda. Kampala: Ministry of Health Uganda; 2020.\n\nKish L: Survey Sampling. John Wiley and Sonsa. New York. 1965; 59–60. Publisher Full Text\n\nOlum R, Chekwech G, Wekha G, et al.: Coronavirus Disease-2019: Knowledge, Attitude, and Practices of Health Care Workers at Makerere University Teaching Hospitals, Uganda. Front Public Health. 2020; 8: 181. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHaridi HK, Al-Ammar AS, Al-Mansour MI: Compliance with infection control standard precautions guidelines: a survey among dental healthcare workers in Hail Region, Saudi Arabia. J Infect Prev. 2016; 17(6): 268–276. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWorld Health Organization: Modes of transmission of virus causing COVID-19: implications for IPC precaution recommendations: scientific brief, 27 March 2020. World Health Organization; 2020. Reference Source\n\nYang M, Wang H, Li Z, et al.: Prevention and Control of COVID-19 Infection in a Chinese Mental Health Center. Front Med (Lausanne). 2020; 7: 356. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGeberemariyam BS, Donka GM, Wordofa B: Assessment of knowledge and practices of healthcare workers towards infection prevention and associated factors in healthcare facilities of West Arsi District, Southeast Ethiopia: a facility-based cross-sectional study. Arch Public Health. 2018; 76: 69. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSahiledengle B, Gebresilassie A, Getahun T, et al.: Infection prevention practices and associated factors among healthcare workers in governmental healthcare facilities in Addis Ababa. Ethiop J Health Sci. 2018; 28(2): 177–86. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRussell D, Dowding DW, McDonald MV, et al.: Factors for compliance with infection control practices in home healthcare: findings from a survey of nurses' knowledge and attitudes toward infection control. Am J Infect Control. 2018; 46(11): 1211–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIbrahim AA, Elshafie SS: Knowledge, awareness, and attitude regarding infection prevention and control among medical students: a call for educational intervention. Adv Med Educ Pract. 2016; 7: 505–10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDesta M, Ayenew T, Sitotaw N, et al.: Knowledge, practice and associated factors of infection prevention among healthcare workers in Debre Markos referral hospital, Northwest Ethiopia. BMC Health Serv Res. 2018; 18(1): 465. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPowell-Jackson T, King JJ, Makungu C, et al.: Infection prevention and control compliance in Tanzanian outpatient facilities: a cross-sectional study with implications for the control of COVID-19. Lancet Glob Health. 2020; 8(6): e780–e789. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBedoya G, Dolinger A, Rogo K, et al.: Observations of infection prevention and control practices in primary health care, Kenya. Bull World Health Organ. 2017; 95(7): 503–516. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWorld Health Organization: Infection prevention and control during health care when COVID-19 is suspected: interim guidance, 19 March 2020. World Health Organization; 2020. Reference Source\n\nCooper S, Wiyeh A, Schmidt BM, et al.: Cochrane corner: factors that influence compliance by healthcare workers with infection prevention and control guidelines for COVID-19 and other respiratory infections. Pan Africa Med J. 2020; 35(23): 23. Publisher Full Text\n\nICN calls for data on healthcare worker infection rates and deaths [press release]. International Council For Nurses. 2020. Reference Source\n\nOchieng MD: More than 1,200 Ugandan health workers infected with COVID-19. CGTN. 2020 November 24, 2020. Reference Source" }	[ { "id": "80019", "date": "09 Mar 2021", "name": "Felix Bongomin", "expertise": [ "Reviewer Expertise Medical Microbiology", "Immunology", "Mycology", "and Internal Medicine." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIt is my pleasure to review this work on Knowledge and Compliance with IPC with regards to COVID-19 prevention among HCWs in 3 major RRHs in Northern Uganda. Patients actively involved in COVID-19 treatment in those centers were excluded and so were those on leave.\nComments to the authors.\nAbstract: Please include the response rates, number of respondents, and basic demographic characteristics in the results section (the 75 could be removed from the methods section).\n\nAbstract: Consider using both % and absolute numbers (n) to express results.\n\nAbstract: Consider adding ORs and 95% to the significant variables to improve interpretation of your results.\n\nIntroduction: \"We believe that HC ...\" could be replaced with appropriate research terms such as We hypothesize...\nMethods Study size: the 69% is not an assumed probably, its knowledge as reported from a previous study among HCWs in Mulago and Makerere.\n\nThe sampling frame is unknown, please clarify.\n\nNumber of respondents (75) throughout the section should be transferred to the results section, the authors are preempting the results within the methods section.\n\nThe response rate should also be transferred to the results section.\n\nPlease add how you compared numerical data (Mann Whitney U, T-test, etc).\n\nInclude how multivariable analysis was done, including 95% CI and OR generation.\nResults Table 2; compliance ; 75-100; ??<75?\n\nTable 3: ORs are not uniform, report to 1 dp for all.\nDiscussion Am surprised the authors started by comparing their results with a Chinese study, when there are numerous Ugandan studies to begin with.\n\nInclude study limitations, strengths and future directions before conclusions.\n\nDo you still think these findings are valid 7 months after the survey?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6494", "date": "12 Apr 2021", "name": "Sharon Bright Amanya", "role": "Author Response", "response": "The reviewer makes a number of important comments and these have been addressed as follows: Comment 1: Abstract: Please include the response rates, number of respondents, and basic demographic characteristics in the results section (the 75 could be removed from the methods section). Revisions have been made in the result section of the abstract to incorporate the suggested information. Comment 2: Abstract: Consider using both % and absolute numbers (n) to express results. Revision made as suggested. Comment 3: Abstract: Consider adding ORs and 95% to the significant variables to improve interpretation of your results. ORs and 95% CI have been added in the results section Comment 4: Introduction: \"We believe that HC ...\" could be replaced with appropriate research terms such as We hypothesize... Revision made as suggested Comment 5: Study size: the 69% is not an assumed probably, its knowledge as reported from a previous study among HCWs in Mulago and Makerere. Revision made as suggested Comment 6: The sampling frame is unknown, please clarify. Sampling frame has been included Comment 7: Number of respondents (75) throughout the section should be transferred to the results section, the authors are preempting the results within the methods section. Number of respondents transferred to the results section Comment 8: The response rate should also be transferred to the results section. Response rate transferred to the results section Comment 9: Please add how you compared numerical data (Mann Whitney U, T-test, etc). Numerical data was such as age and years of work experience were categorized and analyzed as categorical data. Comment 10: Include how multivariable analysis was done, including 95% CI and OR generation. Revision made as requested Comment 11: Table 2; compliance ; 75-100; ??<75? Information has been clarified as follows: Inadequate compliance : 75-100%, inadequate compliance ≤74% of the total compliance score Comment 12: Table 3: ORs are not uniform, report to 1 dp for all. ORs reported in 2dp throughout the table Comment 13: Am surprised the authors started by comparing their results with a Chinese study, when there are numerous Ugandan studies to begin with. Data from Ugandan studies included in the discussion Comment 14: Include study limitations, strengths and future directions before conclusions. Section included Comment 15: Do you still think these findings are valid 7 months after the survey? Indeed there has been a delay in publishing these results since data collection, however the findings are still valid and relevant given the fact that the Covid-19 pandemic is still on-going and the fact that Uganda is usually prone to epidemics that warrant constant adherence to IPC measures." } ] } ]	1	https://f1000research.com/articles/10-136
https://f1000research.com/articles/9-1461/v1	15 Dec 20	{ "type": "Brief Report", "title": "Image analysis method for heterogeneity and porosity characterization of biomimetic hydrogels", "authors": [ "Maryam Jamshidi", "Cavus Falamaki", "Maryam Jamshidi" ], "abstract": "This work presents an image processing procedure for characterization of porosity and heterogeneity of fully hydrated hydrogels based on the analysis of cryogenic scanning electron microscopy (cryo-SEM) images. An algorithm consisting of different filtering, morphological transformation, and thresholding steps to denoise the image whilst emphasizing the hydrogel fibres edges for extracting the pores features is explained. Finally, the information of hydrogel porosity and heterogeneity is presented in form of pore size distribution, spatial contours maps and kernel density dot plots. The obtained results reveal that a non-parametric kernel density plot effectively determines the spatial heterogeneity and porosity of the hydrogel.", "keywords": [ "image processing", "transformation", "noise", "filter", "pixel", "frequency", "kernel", "distribution", "hydrogel", "pore" ], "content": "Introduction\n\nBiomimetic engineered hydrogels often serve as 3D microporous extracellular microenvironment mimics in regenerative medicine, tissue engineering1 and in-vitro cancer studies2. The physical properties of these gels may provide a mechanical cue to regulate the cell phenotypic activities and functions via cellular mechanotransduction3,4. Moreover, it has been shown that a change in the stiffness/elasticity of hydrogel is associated with the morphological change in the structure of the hydrogel mesh5. This article presents an image analysis method to characterise hydrogel structure heterogeneity and porosity based on the cryogenic scanning electron microscopy (cryo-SEM) images of fully hydrated hydrogels.\n\n\nMethods\n\nThe image processing algorithm of the present work has been written as a code in the Python 3.7 language and is able to analyse cryo-SEM images. The present study uses cryo-SEM images of fully hydrated hydrogels adopted with permission from Kaberova et al.6 as input data. To detect the pores precisely, the hydrogel fibre edges are highlighted, and a band pass frequency filter is applied to optimize the removal of noise with preservation of the edges of hydrogel walls.\n\nPre-processing. After loading the image from the specified path, it was first normalized to stretch the Gray level histogram between 0 and 255 and enhance the image contrast. Next, to filter out the noise, a Gaussian (σ =0.7, 3×3) spatial filter convolution was applied to the image. The filtered image is a weighted average of the neighbourhood pixels that better preserve the edges in lower contrast areas while removing the noise7. Afterwards, to extract the edges of the hydrogel fibres, a range of the non-linear 3×3 edge detection filters including Sobel was applied to the image to highlight the locations of sharp intensity transitions. Both the noise and edges belong to the high pass frequencies of the image in essence8 and the edge emphasising filter might introduce some artifacts in these range. Therefore, in the next step, a band pass frequency filter was used to highlight the desired quasi- high range of the frequencies for a better edge detection (refer to Figure 1).\n\nImages on the left show the output images of each step in the processing algorithm and the relevant flowchart is illustrated on the right.\n\nThresholding. The binarization of cryo-SEM images of gels is challenging as there is no standard method for thresholding. Since in most of the cryo-SEM images there is uneven illumination, the adaptive threshold technique was undertaken to segment the background and foreground pores. The algorithm calculates the optimal value of threshold based on the weighted mean and standard deviation of the pixel values within the neighbouring window of fixed size for each pixel and outperforms the conventional methods9. In the present work, the adaptive Gaussian threshold has been applied to the image on a window size of 25×25 pixels. The window size should be optimized depending on the density of the details (information) and a lower or greater size may be chosen.\n\nMorphological transformation. The pores touching the boarders of the image were excluded and then basic morphological transformation (erosion×1 and opening×5) was applied to ensure that the isolated pixels both in the background and foreground (pores) were eliminated. For all transformations, a 3×3 elliptical structuring element was applied. Erosion transformation was applied to separate the touching pores and remove the remaining very small pores. Following, the holes within the detected pores were filled up. And lastly, the image was reconstructed based on the erosion and opening results and then a watershed algorithm was used to segment the pores and measure pore properties on the final image.\n\nTo validate the method, the screenshot images of a hydrogel network from a previously published work where the corresponding pore diameters have been reported, were analysed with the proposed method using OTSU threshold and setting 0.05 max watershed threshold. The obtained results of the proposed method (17 µm) were compared with the available reported measurement (15 µm from Figure 4)10.\n\nAfter performing the pore detection analysis, each single detected pore was associated with the centre of mass and area and the results were exported in a text file. The cryo-SEM image of the sample hydrogel shown in Figure 1A reveals that the hydrogel structure is heterogenous in spatial domain. Therefore, pore size distribution and statistical analysis alone might not represent the spatial heterogeneity and clustering of the detected pores. To quantify and visualize the spatial heterogeneity of the hydrogel, the kernel density estimation function was fitted on the centre of mass of the detected pores on the spatial domain of the cryo-SEM image.\n\n\nResults\n\nComparing the obtained results of the proposed method with the available reported measurements of the pore sizes of fluorescent images of hydrogel elsewhere10, the method has been validated with acceptable accuracy. The results of spatial heterogeneity quantification are presented in the form of contour plots (Figure 2). A higher density value indicates the presence of more pores in the unit of area; therefore, it might represent the location of smaller pore clusters and compactness of the pore clusters. On the other hand, a lower value indicates a less dense area, or an area covered with larger pores. It can be observed from the kernel density contours of a sample image (Figure 2D) that the distribution of most of the larger pores are almost uniformly dispersed; however, the smaller pores formed clusters in the left-hand side and the corner.\n\nA) Detected pores of cryo-SEM micrographs. B) Pore size distribution. C) Spatial contour maps of pore area. D) Kernel density estimation dot plots demonstrating the spatial density of detected pores. Data adapted from Kaberova et al.6 with permission.\n\n\nConclusion\n\nThe algorithm provides an image analysis method for biomaterial science research to investigate the structural heterogeneity of hydrogels. This simple and flexible analysis method allows optimization of different parameters to ideally analyse a broad range of images. We have also demonstrated that based on the data extracted from the image, the kernel density estimation function is a powerful graphical tool to visualize and compare spatial heterogeneity and porosity of the hydrogel. The application of this method can be extended to structural analysis of any fibrous network.\n\n\nData availability\n\nZenodo: niliou/Hydrogel-pore-size: Hydrogel pore size distribution. https://doi.org/10.5281/zenodo.430890711.\n\nThis project contains the following underlying data:\n\n- Hydrogel pore size source images (original source image files in TIF format)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\n\nCode availability\n\nSource code available from: https://github.com/niliou/Hydrogel-pore-size.git\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.430890711.\n\nLicense: MIT Licence", "appendix": "Acknowledgments\n\nThe authors would like to thank Niloufar Jamshidi (PhD candidate, UNSW) for her technical assistance particularly in developing the code. This publication was supported by COST Action NEUBIAS (CA15124), funded by COST (European Cooperation in Science and Technology).\n\n\nReferences\n\nMantha S, Pillai S, Khayambashi P, et al.: Smart Hydrogels in Tissue Engineering and Regenerative Medicine. Materials (Basel). 2019; 12(20): 3323. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPark KM, Lewis D, Gerecht S: Bioinspired Hydrogels to Engineer Cancer Microenvironments. Annu Rev Biomed Eng. 2017; 19: 109–33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCavo M, Fato M, Peñuela L, et al.: Microenvironment complexity and matrix stiffness regulate breast cancer cell activity in a 3D in vitro model. Sci Rep. 2016; 6: 35367. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nd’Angelo M, Benedetti E, Tupone MG, et al.: The Role of Stiffness in Cell Reprogramming: A Potential Role for Biomaterials in Inducing Tissue Regeneration. Cells. 2019; 8(9): 1036. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMarmorat C, Arinstein A, Koifman N, et al.: Cryo-Imaging of Hydrogels Supermolecular Structure. Sci Rep. 2016; 6(1): 25495. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKaberova Z, Karpushkin E, Nevoralová M, et al.: Microscopic Structure of Swollen Hydrogels by Scanning Electron and Light Microscopies: Artifacts and Reality. Polymers (Basel) 2020; 12(3): 578. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBasu M: Gaussian-based edge-detection methods-a survey. IEEE Transactions on Systems, Man, and Cybernetics, Part C (Applications and Reviews). 2002; 32(3): 252–60. Publisher Full Text\n\nEng HL, Ma KK: Noise adaptive soft-switching median filter. IEEE Trans Image Process. 2001; 10(2): 242–51. PubMed Abstract \| Publisher Full Text\n\nCai Z, Ou Y, Ling Y, et al.: Feature Detection and Matching With Linear Adjustment and Adaptive Thresholding. 2020; 8: 189735–46. Publisher Full Text\n\nBodenberger N, Kubiczek D, Abrosimova I, et al.: Evaluation of methods for pore generation and their influence on physio-chemical properties of a protein based hydrogel. Biotechnol Rep (Amst). 2016; 12: 6–12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMaryam, et al.: niliou/Hydrogel-pore-size: Hydrogel pore size distribution (Version R1). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.4308907" }	[ { "id": "79780", "date": "02 Mar 2021", "name": "Miroslava Dušková-Smrčková", "expertise": [ "Reviewer Expertise macromolecular chemistry" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIt brings a useful piece of computer image analyses software development applicable to quantification of images of heterogeneous materials, namely of 2D projection of pores/distinguishable distributed objects – such as bubbles, particles etc.\nFirst, a brief generic remark. Presently, a number of image analysis software packages for materials science, commercial as well as open-source, for complex scientific image processing are available. However, it is an increasing challenge for the end user to understand the routine behind the image processing and 2D or 3D structure rendering and sometimes the description of the certain function under “Help” does not offer much possibility to grasp the principle.\nThe presented work is particularly concentrated on the analysis of microscopy images of macroporous hydrogels where the advancement of image processing such as the proposed method is still needed: the reason is that we need to investigate relations between hydrogel morphology, microstructure and hydrogel macroscopic properties, mainly mechanical behavior and its effects in biological application – because not everything is known and there are new types of hydrogels of plethora structures being introduced by chemists.\nOn top of that, besides the scientific paper containing detailed explanation of itself, Jamshidi and Falamaki made their script (written in Python) available to scientific community – that I found really useful. With more users, we can expect evaluation of high number of samples and possibly building up the software package for more versatile gel characterization. Because of detailed explanation of the hydrogel image processing, the work offers possibility to other interested researchers on the border areas between hydrogel (materials) design and image processing to learn and extend the work. Therefore, I consider publishing this paper with F1000 Research valuable.\nBefore indexing the manuscript, I still propose some modification and clarification of few points of discussion – mainly to make the paper speaking to hydrogel designers and chemists who are most of the time users or advanced users but do not have the ambition to be software developers.\n\nA very important point to clarify is the very first sentence of the Abstract. The authors say: “This work presents an image processing procedure for characterization of porosity and heterogeneity of fully hydrated hydrogels based on the analysis of cryogenic scanning electron microscopy (cryo-SEM) images.” Let’s make this clear: analysis of cryo-SEM images may but may not reveal the structure as in hydrated state. When a fully hydrated hydrogel sample is processed for cryogenic EM, it goes through an inevitable freezing step. The morphology of hydrated sample most likely will be altered depending on the conditions of the freezing step but also depending on the particular sample. Freezing, whatever rapid, will cause change in volume and shape of existing pores1, possibly will cause formation of new pores, or will cause some failure, cracks, distortion of the gel phase. Or it will just cause isotropic shrinking of the hydrogel volume. We can’t exactly predict the change. This means that whatever structure of gel the cryo-SEM reveals, it may not correspond to the structure in fully hydrated – meaning swollen in water to equilibrium under given conditions – hydrogel. Only in rather special cases, for example in less swollen and mechanically stronger gels, the gel matrix can resist changes, or in the case of macroscopic gels with arrangement of connected spheres (see Fig. 4 of paper by Kalasova et al in DOI 10.1109/TIM.2020.29952322) it can just isotropically contract (as shown in already cited work (https://doi.org/10.3390/polym120305783). This all said in the above paragraph does not disrate the impact of the developed image processing – as it can be well applied to images obtained by other methods, too. This point indeed, should be taken into account in the introductory part – pg. 3, where the text again reads: “This article presents an image analysis method to characterise hydrogel structure heterogeneity and porosity based on the cryogenic scanning electron microscopy (cryo-SEM) images of fully hydrated hydrogels.” I propose to modify the formulation similarly as: “This article presents an image analysis method to characterise hydrogel structure heterogeneity and porosity resulting from the treatment of fully hydrated hydrogels – plunge-freezing of hydrated samples done to acquire their cryogenic scanning electron microscopy (cryo-SEM) images.”\n\nSeveral times authors speak about “hydrogel fibers” – I wish to bring up that the form of hydrogel is not fibrous, the gel forms rather thin walls separating the pores in 3D space.\n\nThere are background and foreground pores mentioned – pls add some schematics what that exactly means and where the reader can see the background vs. the foreground pores on the presented figures.\n\nWhat does the “OTSU” abbreviation stand for and what is OTSU threshold mentioned in part “Morphological transformation”? (abbreviation not explained)\n\nThe reference where from the Figure 1 was adopted should be given also in the Figure 1 legend.\n\nThe size of the figures and their resolution even in supplied pptx files is both quite low. Especially the small size of printed letters in the images is critical and must be increased (and improved resolution): legend, axis descriptions, numbers within the Figure 2 are not readable. There are two images both designated Fig.1/ B.\n\nThe actual results shown on Figure 2 – mean pore size, pore size distribution …is not discussed in numbers. It should also be compared with the original evaluation given in the source article. To determine the size of pores should be the target of the presented method.\n\nIt would be of interest to discuss the existing limits of the presented method: for example how the method will tackle anisotropic pores, either regularly distributed or clustered or even oriented? – Will the method detect connecting pores, etc? Indeed, this can be the future direction but even then will be worthwhile mentioning.\n\nIt would be of interest to adopt the method to evaluate porosity: the void volume fraction in the sample.\n\nThe thickness of the gel wall separating pores is also of interest and should be also analysed by IA package.\n\nLast but not least my comment of Conclusion: The material depicted on Figures 1 and 2 should not be called fibrous network – the pores in it look like those in the sponge for washing dishes: pores surrounded by thinner or thicker hydrogel walls of thickness typically 100-101 um. Indeed, the molecular basis of this material is macromolecular network, that means that all building units molecules are connected to one molecule of a 3D macromolecular network spanning the whole sample space but this molecular nature of the samples indeed is far below resolution of currently available visualization methods.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6505", "date": "12 Apr 2021", "name": "Cavus Falamaki", "role": "Author Response", "response": "Author’s Respond to Reviewer: Authors would like to sincerely thank reviewer not only for their constructive comments and scientific clarification that improves the scientific quality of the paper, but also for their suggestions that can be used to further develop the current piece of work. All modifications made in the manuscript are shown with . Reply to reviewers’ comments are shown with #. It brings a useful piece of computer image analyses software development applicable to quantification of images of heterogeneous materials, namely of 2D projection of pores/distinguishable distributed objects – such as bubbles, particles etc. First, a brief generic remark. Presently, a number of image analysis software packages for materials science, commercial as well as open-source, for complex scientific image processing are available. However, it is an increasing challenge for the end user to understand the routine behind the image processing and 2D or 3D structure rendering and sometimes the description of the certain function under “Help” does not offer much possibility to grasp the principle. The presented work is particularly concentrated on the analysis of microscopy images of macroporous hydrogels where the advancement of image processing such as the proposed method is still needed: the reason is that we need to investigate relations between hydrogel morphology, microstructure and hydrogel macroscopic properties, mainly mechanical behavior and its effects in biological application – because not everything is known and there are new types of hydrogels of plethora structures being introduced by chemists. On top of that, besides the scientific paper containing detailed explanation of itself, Jamshidi and Falamaki made their script (written in Python) available to scientific community – that I found really useful. With more users, we can expect evaluation of high number of samples and possibly building up the software package for more versatile gel characterization. Because of detailed explanation of the hydrogel image processing, the work offers possibility to other interested researchers on the border areas between hydrogel (materials) design and image processing to learn and extend the work. Therefore, I consider publishing this paper with F1000 Research valuable. Before indexing the manuscript, I still propose some modification and clarification of few points of discussion – mainly to make the paper speaking to hydrogel designers and chemists who are most of the time users or advanced users but do not have the ambition to be software developers. A very important point to clarify is the very first sentence of the Abstract. The authors say: “This work presents an image processing procedure for characterization of porosity and heterogeneity of fully hydrated hydrogels based on the analysis of cryogenic scanning electron microscopy (cryo-SEM) images.” Let’s make this clear: analysis of cryo-SEM images may but may not reveal the structure as in hydrated state. When a fully hydrated hydrogel sample is processed for cryogenic EM, it goes through an inevitable freezing step. The morphology of hydrated sample most likely will be altered depending on the conditions of the freezing step but also depending on the particular sample. Freezing, whatever rapid, will cause change in volume and shape of existing pores1, possibly will cause formation of new pores, or will cause some failure, cracks, distortion of the gel phase. Or it will just cause isotropic shrinking of the hydrogel volume. We can’t exactly predict the change. This means that whatever structure of gel the cryo-SEM reveals, it may not correspond to the structure in fully hydrated – meaning swollen in water to equilibrium under given conditions – hydrogel. Only in rather special cases, for example in less swollen and mechanically stronger gels, the gel matrix can resist changes, or in the case of macroscopic gels with arrangement of connected spheres (see Fig. 4 of paper by Kalasova et al in DOI 10.1109/TIM.2020.29952322) it can just isotropically contract (as shown in already cited work (https://doi.org/10.3390/polym120305783). This all said in the above paragraph does not disrate the impact of the developed image processing – as it can be well applied to images obtained by other methods, too. This point indeed, should be taken into account in the introductory part – pg. 3, where the text again reads: “This article presents an image analysis method to characterise hydrogel structure heterogeneity and porosity based on the cryogenic scanning electron microscopy (cryo-SEM) images of fully hydrated hydrogels.” I propose to modify the formulation similarly as: “This article presents an image analysis method to characterise hydrogel structure heterogeneity and porosity resulting from the treatment of fully hydrated hydrogels – plunge-freezing of hydrated samples done to acquire their cryogenic scanning electron microscopy (cryo-SEM) images.” # The authors agree and admit that in the paper it should be clarified that there are some artifacts associated with the imaging methods specially cryo-SEM and proper referencing should be made. # The abstract is revised accordingly, and some clarifications is added to the introduction part to cover the reviewer’s points: This work presents an image processing procedure for characterization of porosity and heterogeneity of hydrogels network mainly based on the analysis of cryogenic scanning electron microscopy (cryo-SEM) images and can be extended to any other type of microscopy images of hydrogel porous network. #The following paragraph is modified in the introduction part: This article presents an image analysis method applied to characterise hydrogel structure heterogeneity and porosity resulting from the treatment of fully hydrated hydrogels during plunge-freezing to acquire their cryogenic scanning electron microscopy (cryo-SEM) images. It should be mentioned that during the process of sample preparation for cryo-SEM microscopy, the sample undergoes a probable morphological alteration, furthermore the process itself might introduce artifacts according to the nature of the material and swelling rate. Readers are encouraged to refer to the studies specialized on the imaging of different hydrogel network by Kalasova et. al 1 and Pradny et. al2. Several times authors speak about “hydrogel fibers” – I wish to bring up that the form of hydrogel is not fibrous, the gel forms rather thin walls separating the pores in 3D space. # Authors acknowledge the point, in the manuscript wherever the term “fibre” is used, replaced with “hydrogel walls”. There are background and foreground pores mentioned – pls add some schematics what that exactly means and where the reader can see the background vs. the foreground pores on the presented figures. # Authors agree that there is a need to clarify the terms used to explain the method. The image of the hydrogel is first segmented into two categories of the hydrogel walls and pores. Because in this analysis we aim to detect the pores, therefore the pores segment is foreground (value 1 – white), and the walls are considered as background (value 0- black). Accordingly, the figure 1 has been edited and a small schematic showing foreground and background is added to the figure 1. # Following section has been corrected also for more clarification in “Thresholding” section and also a schematic is added to Figure 1: Since in most of the cryo-SEM images there is uneven illumination, the adaptive threshold technique was undertaken to segment the background (hydrogel walls, value=0 black) and foreground pores (pores, value=1 white). Figure 1. Image processing algorithm of sample cryo-SEM micrograph of glycidyl methacrylate hydrogel crosslinked with 0.3 mol% (ethylene glycol) dimethacrylate. Images on the left show the output images of each step in the processing algorithm and the relevant flowchart is illustrated on the right to distinguish foreground (pores) from background (hydrogel walls). The source image adapted from Kaberova et al. 3 with permission. What does the “OTSU” abbreviation stand for and what is OTSU threshold mentioned in part “Morphological transformation”? (abbreviation not explained) # There was a typo here, Otsu’s method is one of the methods used for image binarization or thresholding. The following sentence in under “Morphological transformation” is edited: To validate the method, the screenshot images of a hydrogel network from a previously published work where the corresponding pore diameters have been reported, were analysed with the proposed method using Otsu’s thresholding method and setting 0.05 max watershed threshold. # in the corresponding python script under thresholding section, both methods of binarization (Otsu’s and Adaptive are provided), user can comment each line and use what is best to process their image. We have used Otsu’s for validation, and adaptive method is suggested for cryo-EM images. The reference where from the Figure 1 was adopted should be given also in the Figure 1 legend. # The comment has been considered and the legend for figure 1 is revised as bellow: Figure 1. Image processing algorithm of sample cryo-SEM micrograph of glycidyl methacrylate hydrogel crosslinked with 0.3 mol% (ethylene glycol) dimethacrylate. Images on the left show the output images of each step in the processing algorithm and the relevant flowchart is illustrated on the right to distinguish foreground (pores) from background (hydrogel walls). The source image adapted from Kaberova et al. 3 with permission. The size of the figures and their resolution even in supplied pptx files is both quite low. Especially the small size of printed letters in the images is critical and must be increased (and improved resolution): legend, axis descriptions, numbers within the Figure 2 are not readable. There are two images both designated Fig.1/ B. #The comment considered, the resolution of the figures increased and high-quality images in actual sizes has been provided in power point files as attachment. The actual results shown on Figure 2 – mean pore size, pore size distribution …is not discussed in numbers. It should also be compared with the original evaluation given in the source article. To determine the size of pores should be the target of the presented method. # Authors consider the comment; however, it should be noted that for this paper we have analysed only one image (Figure 1. e) of the source article by Kaberova et. al 3. This image is related to sample G0/0.3 in the source article which the pore size is reported between 2-40 µm (we assume that these values are based on equivalent diameter) for this sample according to Table 1. Using the current method, the average equivalent diameter of the detected pores is equal to 9.45 pixels or 12.36 µm (pixel to microns scale of the image = 500 µm/ 382 pixel). The actual pore size distribution is updated in Figure 2, from this image it is apparent that the measured pore size distribution by this method sit between 2 – 36 µm. # the correction applied to the Results section of the manuscript: Moreover, the average equivalent diameter of the pores of the analysed source image (adopted from Kaberova et. al 3) was equal to 12.36 µm with the corresponding pore size distribution range of 2-36 µm. The comparison of the results with the range reported by Kaberova et al.3 (2–40 µm) shows a good agreement and reliability of the presented method as it is shown in Figure 2B. It would be of interest to discuss the existing limits of the presented method: for example how the method will tackle anisotropic pores, either regularly distributed or clustered or even oriented? – Will the method detect connecting pores, etc? Indeed, this can be the future direction but even then will be worthwhile mentioning. # This is a very interesting point picked up by reviewer. Authors clarify that the current method does not classify the hydrogel network as “clustered” or “distributed” and we have not done any statistical analysis to focus on quantification of anisotropic properties of the hydrogel network. However, structural and cluster analysis of the gel network is the topic of the next paper currently developing by one of the team members. The result and relevant script of the next work will be available to scientific community after publishing. # For the detection of the connecting pores, we have applied watershed transform which is a well-established method to separate touching objects, however authors admit that there are more advanced and complex methods/ pipelines to segment the pores or hydrogel walls. The might be of interest to apply a recently developed methods that combines the classic “watershed” segmentation with “deep learning” 4 to increase the level of accuracy and performance of the segmentation method of the analysis package. # The following sentences under Conclusion part is revised in the manuscript: The application of this method can be extended to structural analysis of any other porous network. Furthermore, it is worth mentioning that applying more sophisticated segmentation methods4 to combine classical transformation with deep learning models in the future works might improve the accuracy and performance of the method to distinguish touching pores and separate hydrogel walls. It would be of interest to adopt the method to evaluate porosity: the void volume fraction in the sample. # Authors acknowledge the reviewer’s point. The void fraction calculation has appended to the script and it is printed in the console when the script run has ended (open images should be closed). We consider 2D void fraction = total areal of pores / image area. # Script new version (R2 is uploaded on github accordingly). https://github.com/niliou/Hydrogel-pore-size/blob/main/Hydrogel%20Pore%20Size%20Distribution%20-%20R2%20Final.py The thickness of the gel wall separating pores is also of interest and should be also analysed by IA package. # We admit the comment, the present method can also be used to measure wall thickness of the gel. The only change to be made is the background and fore ground that should be reversed (fore ground is of interest = white) instead of pores we need to bring walls to the front or white. Another point is that for thresholding some values should be tweaked that is commented in the corresponding section in the script. Therefore, by inversing the image look up table the wall thickness can be measured. For the source image we have: # should be noted that in the results file the minimum axis value should be considered as closet parameter describing hydrogel walls. The value of the average wall thickness for the source image is equal to 4.79 pixels or 6.26 µm. # The following clarification has been made to the Image processing section: To detect and measure the thickness of the hydrogel walls, the look up table of the binary image should be inversed to bring hydrogel walls in foreground (white). #The relevant hints are also commented in the script. #To invert the look-up table of the image in the first part, Then, line 130 should be uncommented and used for hydrogel wall thickness measurement, the input image in the command line (130) could be any of the filtered images that we have included in the script, so the end user can choose the best filter that gives the best results, in the above we have chosen “DoGNN” image which is filtered image by Difference of Gaussian method. In the script we have several filters (lines 45-84) + bandpass filter lines (103-104) that mainly focuses on edge feature detection. We have commented in the script describing what filters are applied in each section. So, user can choose which one of these filters can extract the wall features the best. Line 144 also can be commented in case “hydrogel wall thickness” is being measured. Last but not least my comment of Conclusion: The material depicted on Figures 1 and 2 should not be called fibrous network – the pores in it look like those in the sponge for washing dishes: pores surrounded by thinner or thicker hydrogel walls of thickness typically 100-101 um. Indeed, the molecular basis of this material is macromolecular network, that means that all building units molecules are connected to one molecule of a 3D macromolecular network spanning the whole sample space but this molecular nature of the samples indeed is far below resolution of currently available visualization methods. # Authors appreciate reviewer’s attention and their explanation to improve the scientific value of the work as well as authors understanding of the nature of hydrogel network. Anywhere in the manuscript the term “fibrous” is revised and replaced. 1. Kalasová D, Břínek A, Šlouf M, Dušková-Smrčková M, Zikmund T, Patáková Z, et al. Wide-Cone Angle Phase-Contrast X-Ray Computed Tomography of Synthetic Polymer Materials. IEEE Transactions on Instrumentation and Measurement. 2020; 69(11):8910-8. 2. Přádný M, Dušková-Smrčková M, Dušek K, Janoušková O, Sadakbayeva Z, Šlouf M, et al. Macroporous 2-hydroxyethyl methacrylate hydrogels of dual porosity for cell cultivation: morphology, swelling, permeability, and mechanical behavior. 2014; 21(11):1-12. 3. Kaberova Z, Karpushkin E, Nevoralová M, Vetrík M, Šlouf M, Dušková-Smrčková M. Microscopic Structure of Swollen Hydrogels by Scanning Electron and Light Microscopies: Artifacts and Reality. Polymers. 2020; 12(3):578. 4. Bai M, Urtasun R, editors. Deep watershed transform for instance segmentation. 2017." } ] } ]	1	https://f1000research.com/articles/9-1461
https://f1000research.com/articles/10-284/v1	12 Apr 21	{ "type": "Research Article", "title": "Use of the bar chart/S-curve and computerized precedence diagram method on scheduling and controlling building construction projects by contractors: a cross-sectional study", "authors": [ "Putri Lynna Adelinna Luthan", "Nathanael Sitanggang", "Abdul Hamid", "Bambang Hadibroto", "Nathanael Sitanggang", "Abdul Hamid", "Bambang Hadibroto" ], "abstract": "Background: Building construction projects have very complex activities, so they require precise and accurate methods of scheduling and control. Using the right method, the project executor can carry out the project according to plan and any schedule deviations can be controlled effectively. This study aims to compare the effectiveness of using the bar chart/S-curve and computerized precedence diagram method (PDM) on scheduling and controlling building construction projects. Methods: The use of the two methods and their effectiveness during project work were analysed using a survey directed to building construction workers. Results: A total of 50 workers completed the survey. The use of PDM (using Microsoft Project) was significantly more effective than the bar chart/S-curve method in scheduling building construction projects (t count 15.516> t table 2.660) and controlling building construction projects (t count 17.233> t table 2.660). In addition, PDM was associated with allowing the project to find the critical path more quickly, overcoming project delays more effectively. Conclusions: By using PDM, a on a building construction project’s schedule for the implementation of the work can be changed immediately, if there is a delay or deviation of work. The findings of this study are useful for construction service companies and the development of construction management science in civil engineering study programs.", "keywords": [ "S-curve", "PDM", "scheduling", "control" ], "content": "Introduction\n\nCost, quality and time are the three main indicators that determine the success of a construction project. A construction project is successful if the costs incurred are in accordance with the budget, the time for completion is in accordance with the scheduled time, and the resulting quality is as planned. This is consistent with the description by Soemardi et al.,1 who stated that cost and time are among the keys to project success. The successful implementation of a project is inseparable from the effectiveness of the scheduling and controlling, which provides accurate information about the schedule for the work plan to be carried out and the actions that need to be taken if deviation from the project occurs. The purpose of scheduling is to determine the sequence of activities to be carried out, the dependency relationship of each activity, the resource requirements of each activity and the allocation of expected implementation time. With the development of information technology, construction managers are starting to look for suitable applications to make it easier to plan and control a project so that it is on time, on budget and meets the required quality. Many programs are offered to process data related to controlling a project. These programs make it easier for construction workers to input the work that has been carried out. The increasing demand for the acceleration of work schedules has led to the development of computerized planning and scheduling.2\n\nPandey et al.4 stated that delay in a project is due to the use of inaccurate information in preparing work schedules. Memon et al.5 ascribed delay to incorrect planning and scheduling by the construction manager, while ineffective scheduling and control were the reasons given by Pourrostam and Ismail.6 Further, according to Odeh and Battaineh,7 improper planning was responsible, while inadequate planning and scheduling were the causes according to Romuald-Kokou et al.8 Therefore, project planning requires a precise and accurate method.\n\nToday’s computer application programs greatly assist construction managers in entering project data, managing project activities and people, project control, and project reports. Harris9 describes a project as a set of operations or activities that must be planned and arranged in a logical order to achieve a determined outcome at a definitive end time. Before the schedule is drawn up, the project manager plans the manpower that will be involved in the work, which is outlined through a hierarchy known as the work breakdown structure. Several scheduling methods that are often used by construction managers include bar charts, S-curves, and network methods, such as program evaluation and review technique (PERT), critical path method (CPM) and precedence diagram method (PDM). The PDM is now being used in the field because it is assisted by computer applications. Although the PDM can be computerized with Microsoft (MS) Project, many construction managers still use the S-curve as a scheduling and control tool.3\n\nA bar chart is a set of activities arranged vertically and a time scale arranged horizontally based on the length of the bar chart, which means that the beginning of the bar indicates the start of work and the end signifies work completion. A bar chart is always accompanied by an S-curve, which is arranged based on the budget and completion time of each task. The S-curve can show the progress of the project based on activities, time, and work weight represented as a cumulative percentage of all project activities. Figure 1 shows two curves that have different meanings, namely the work plan and the actual work. In Figure 1, it is shown that the project was delayed, did not go according to plan and underwent deviations. According to Luthan and Sitanggang,10 the mismatch between plan and implementation is shown by the S-curve as the magnitude of the deviation that occurs.\n\nThis figure has been reproduced with permission from Luthan, P.L.A. & Sitanggang, N. (2016). Penerapan Earned Value pada Aplikasi MS. Project Sebagai Pengendali Proyek (Studi Kasus Pada Proyek di kota Medan), Prosiding Konferensi Nasional Teknik Sipil 10, Universitas Atmajaya Yogyakarta, 26-27 October.\n\nThe bar chart and S-curve method is used to bid for projects by contractors. The S-curve is used as a control tool based on the costs incurred by each work item using MS Excel to input the actual data on the bar chart, as shown in Figure 1 on the implementation curve, so that the construction manager will know that the project has been delayed by 22%.\n\nMeanwhile, MS Project is an application developed based on a network method, namely PDM, which is structured to make a comprehensive schedule that can determine the critical path.11 On MS Project, the project will show a bar chart and the dependency relationship of each activity; this depiction is not different from the S-curve/bar chart method. The relationship between two activities in PDM consists of four varying types: (1) Finish to finish, (2) Finish to start, (3) Start to start, (4) Start to finish.\n\nHermawan12 explained that large-scale projects with numerous activities can no longer be controlled manually, so an application that can detect problems in the field is needed. MS Project-PDM is an application that can be used in the field for scheduling and control. According to Harris,9 there are four stages/levels in planning and scheduling: planning without people; monitoring progress without people; scheduling with people, roles, and budget; and monitoring the progress of a resource schedule. For details, see Table 1.\n\nMS Project-PDM is used to determine the critical path to completing the activity immediately; if it is not completed within the specified time, it will affect other activities. According to Krajewski,13 determining the critical path is based on the network planning method to estimate the right time to carry out and end an activity. Furthermore, Bansal and Pal14 stated that the linkage of activities in the critical path using a 3-dimensional model will show an easy-to-understand sequence of activities, so that construction managers can determine the actions to be taken. Sabariah et al.3 found that the use of the PDM is still relatively low. Based on the description above, an important question arises: between the bar chart/S-curve and MS Project-PDM methods, which one performs better in scheduling and controlling of building construction projects? In this study, a comparison of the performance of the two methods was done. The findings provide useful information for construction companies and the development of construction management science in civil engineering.\n\n\nMethods\n\nThis study used a cross-sectional survey method design, and was carried out from March to April 2020.\n\nPurposive sampling was used to select workers in building construction. Participants were approached through collaboration with the Indonesian Project Scheduling Expert Association (PAPPI); the researcher contacted PAPPI to distribute questionnaires to participants. Inclusion criteria included workers who had attended training in the use of the MS Project application (PDM) organized by the Indonesian Project Scheduling Experts Association. A total of 50 PAPPI members fit the criteria needed to fill out the questionnaire.\n\nTo collect data, a survey was sent online using the Google Form application (see Extended data16). The survey included various statements (see Table 2) and respondents were asked to response to each statement with the following options: STS, strongly disagree; TS, disagree; N, neutral; S, agree; SS, strongly agree.\n\nInstrument testing was carried out on three workers prior to the study. This was done to determine the readability of the instrument, the clarity of the content in each statement, and the language of the instrument. After the test was conducted, it was concluded that all statement items had a good readability level, contained clear content, and the language used was easy to understand. The instrument reliability coefficient of using the S-curve was 0.98, and the instrument reliability coefficient of using the MS Project-PDM was 0.96.\n\nDescriptive analysis and a t-test (Sugiyono15) were carried out for data analysis using Microsoft Excel 2010. Descriptive analysis was used to determine the distribution of answers from respondents which in turn described the use of S-curve and MS Project for scheduling using the S-and supervision. A t-test was used to test the significant differences in scheduling and supervision between the use of the S-curve and MS Project.\n\nEthical approval was not sought for this study due to the low risk nature of the survey and study population. At the beginning of the Google Form, information about the study, data collection and data use was presented to participants. Completion of the survey was taken as consent to participate in the study.\n\n\nResults and discussion\n\nA total of 50 workers responded to the survey. Based on work experience as project scheduling experts, 28% of participants had experience of less than 5 years, 20% had 5-10 years of experience, 18% had 10-15 years of experience, and most, 34%, had experience over 15 years. The majority of participants were male (n = 47).\n\nTable 2 shows the ten statements distributed to respondents for each method of scheduling and control.\n\nRegarding the distribution of answers to the ten questions on the use of S Curve-Bar Chart in project scheduling, 47.92% of respondents chose ‘strongly disagree’, while 45.83% chose ‘disagree’. Very few respondents chose ‘strongly agree’, ranging from 6.25-10.42%. Regarding the responses to questions about the use of the S-curve/bar chart in project scheduling, the largest number of respondents chose ‘strongly disagree’ (47.92%), that is, the S curve-bar chart cannot show critical work. ‘Strongly agree’ was chosen by the least number of respondents (6.25%), that is, when making a bar chart schedule, the S-curve does not need to be done in detail; this option was not chosen by experienced people. Regarding respondents' answers to scheduling with the use of S-curve/bar charts, 43.75% of respondents chose ‘agree’ or ‘strongly agree’, that is, construction managers still use S-curve/bar chart as a tool to make project bids.\n\nSTS, strongly disagree; TS, disagree; N, neutral; S, agree; SS, strongly agree.\n\nRegarding the distribution of answers to ten questions on the use of the MS Project-PDM in project scheduling, respondents dominantly chose ‘strongly agree’ (81.25%), while ‘strongly disagree’ recorded 0%. Very few respondents chose ‘disagree’, ranging from 4.17-10.42%. Concerning responses to questions about the use of the MS Project-PDM method in project scheduling, the largest number of respondents chose ‘strongly agree’ (81.25%), while no respondents chose ‘strongly disagree’ (0%).\n\nSTS, strongly disagree; TS, disagree; N, neutral; S, agree; SS, strongly agree.\n\nWith respect to the distribution of answers to ten questions on using S-curve/bar chart for project control, respondents dominantly chose ‘disagree’ (52.08%). Very few respondents chose ‘strongly agree’, ranging from 6.25-8.33%. Regarding the responses to questions about the use of the S-curve/bar chart method in project control, the largest number of respondents chose ‘agree’ (52.08%), that is, the S-curve/bar chart cannot be used as a strategy to reduce delay. The lowest number of respondents chose the ‘strongly disagree’ option (6.25%), in the categories of X35, X36, X37, X38, X39, and X310 (see Table 2).\n\nSTS, strongly disagree; TS, disagree; N, neutral; S, agree; SS, strongly agree.\n\nConcerning the distribution of answers to ten questions on using the MS Project-PDM method for project control, respondents dominantly chose ‘strongly agree’ (62.50%), while ‘strongly disagree’ recorded 0%. Very few respondents chose ‘disagree’, ranging from 4.17-6.25%. With respect to responses to questions about the use of the MS Project-PDM in project control, the largest number of respondents (62.50%) chose the ‘strongly disagree’ option for X48, while none chose ‘strongly agree’ (0%).\n\nSTS, strongly disagree; TS, disagree; N, neutral; S, agree; SS, strongly agree.\n\nSome construction managers still use S-curve/bar chart as a tool to bid for projects (43.75%). However, in the future, S-curve/bar chart would no longer be used as a tool for project scheduling. Moreover, regarding the use of MS Project-PDM in scheduling, most respondents agreed with the questions, but there were questions that had the highest number of positive responses, including X29 (81.25%) and X28 (75%). X29 states that in the MS Project-PDM scheduling, the critical path is known. This is very important for construction managers because the critical path is a path whose activities need attention; if the activity on the critical path is delayed, it will affect the next activity and the project will experience delays. X28 states that the MS Project-PDM scheduling will show activities that have free time (free float), which means that even though the assigned duration of the activity is 5 days, if it is not completed within 5 days, the project will not experience delays. However, on the S-curve/bar chart, if the work time is not as scheduled, the project will experience delays.\n\nThe t value is 15.516> t table, 2,660. Therefore, the observed mean difference (mean value) is significant at 1% significance level. Thus, the use of MS Project-PDM is more effective than the S-curve/bar chart method in scheduling building construction projects.\n\nRegarding the use of S-curve/bar chart in controlling projects, very few respondents agreed that it can be used as a control tool. Of the ten questions, 37.50% to 52.08% of respondents chose the ‘disagree’ option, while 6.25% to 8.33% chose the ‘strongly agree’ option, meaning that the respondents actually answered that the S-curve/bar chart cannot be used as a control tool in a construction project. However, concerning the MS Project-PDM, 52.08-62.50% of respondents chose ‘strongly agree’, while 4.17-6.25% chose ‘disagree’. This means that MS Project-PDM is an accurate tool for project control according to the respondents, who stated that it is the right tool for carrying out strategies and making decisions in case of delays. In addition, this method can quickly change the schedule if there is a delay or deviation of work, and it can also detect or control the costs used in completing a task.\n\nThe t value is 17.233> t table, 2.660. Therefore, the observed mean difference (mean value) is significant at 1% significance level. Thus, the use of MS Project-PDM method is more effective than the S-curve/bar chart method in controlling building construction projects.\n\n\nConclusions\n\nThis study concludes that the use of the MS Project-PDM is more effective than the S-curve/bar chart method in scheduling and controlling building construction projects. Knowing the critical path is needed by construction managers so that work delays do not occur. The critical path will be known if the construction manager uses MS Project-PDM in scheduling. Furthermore, the use of MS Project-PDM is effective in controlling delays and work deviations that occur; the schedule can be quickly changed so that work delays are resolved properly.\n\n\nData availability\n\nFigshare: Supplementary Data for Comparative Study on the Use of S Curve-Bar Chart and Ms Project-PDM Methods in Scheduling and Controlling Building Construction Project, https://doi.org/10.6084/m9.figshare.14059202.v2.16\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nThanks are conveyed to the Chancellor of Universitas Negeri Medan, who provided the research funds, and the Perhimpunan Ahli Penjadwalan Proyek Indonesia (PAPPI), which facilitated the research so that it can be carried out properly.\n\n\nReferences\n\nSoemardi BW, Wirahadikusumah RD, Abduh M, et al.: Konsep Earned Value untuk Pengelolaan Proyek Konstruksi. Laporan Hasil Riset. ITB; 2006.\n\nNaik MG, Aditya M, Naik SB: GIS Based 4D Model Development for Planning and Scheduling of a Construction Project. Int J Innov. Manag. Technol. 2011; 2(6, December 2011).\n\nSabariah I; dkk: Analisis Metode Network Planning dan S-Curve Proyek Konstruksi di Bogor. Jurnal Astonjadro. 2012; 1(1). Publisher Full Text\n\nPandey RD, Sompie BF, Tarore H: Analisis Faktor Penyebab Pembengkakan Biaya (Cost Overrun) Peralatan pada Proyek Konstruksi Dermaga di Sulawesi Utara. Jurnal Ilmiah Media Eng. 2012; 2(3).\n\nMemon AH, Rahman IA, Abdullah MH, et al.: Factors Affecting Construct ion Cost in Mara Large Construction Project: Perspective of Project Management Consultant. Int J Sustainable Construct. Eng. Tech. 2010; 1(2).\n\nPourrostam T, Ismail A: Significant factors causing and effects of delay in Iranian construction projects. Aust. J Basic Appl. Sci. 2011; 5(7): 450–456. 1991-8178.\n\nOdeh AM dan Battaineh HT:Causes of construction delay: traditional contracts. Int J Project Manag. 2002; 20: 67–73. Publisher Full Text\n\nAkogbe R-KTM, Xin F, Jing Z: Importance and ranking evaluation of delay factors for development construction projects in Benin, KSCE. J Civil Eng. 2013; 17: 1213–1222.\n\nHarris PE: Calculation Differences When Importing From Microsoft Project 2003-2010. Into Oracle Primavera P6 Version 7. 2010; Melbourne:Eastwood Harris Pty Ltd.\n\nLuthan PLA, Sitanggang N: Penerapan Earned Value pada Aplikasi MS. Project Sebagai Pengendali Proyek (Studi Kasus Pada Proyek di kota Medan). Prosiding Konferensi Nasional Teknik Sipil. 10, Universitas Atmajaya Yogyakarta:2016 26-27 October.\n\nMubarok F, Rizal MC, Arumsari N: Optimasi Proyek Filter Water Supply Dengan Metode Precedence Diagram Method-Least Cost Analysis Dengan Penambahan Tenaga Kerja.Surabaya; 2017.\n\nHermawan A: Penggunaan Perangkat Lunak dalam Pengelolaan Proyek Konstruksi. Jurnal Teknik Sipil Fakultas Teknik Unika Soegijapranata. 2006; volume III, No. 1, 1–7.\n\nKrajewski LJ, Malhotra LPR, dan Manoj KM: Operation Management. New Jersey:Pearson; 2010.\n\nBansal VK, Pal M: Potential of geographic information systems in building cost estimation and visualisation. Autom. Constr. 2007; 16(3): 311–322.\n\nSugiyono: Metode Penelitian Pendidikan Pendekatan Kuantitatif, Kualitatif, dan R & D. Bandung:Penerbit Alfabeta.2008.\n\nLuthan PLA, Sitanggang N, Hamid A, et al.: Supplementary Data for Comparative Study on the Use of S Curve-Bar Chart and Ms Project-Pdm Methods in Scheduling and Controlling Building Construction Project. figshare. Dataset. 2021. Publisher Full Text" }	[ { "id": "83159", "date": "19 Apr 2021", "name": "S. M. Reza Alavipour", "expertise": [ "Reviewer Expertise Construction management", "construction scheduling", "construction finance", "construction contracts", "construction financial management" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis research intends to show that PDM has more benefit and is more effective while used in the projects compared to S-Curve or bar chart.\nHowever, this reviewer has the following concerns.\n1) Title:\nThe title is very long and the contribution and originality of the research could not be understood by reading the current title.\n2) Introduction:\nThere are many more research studies that proposed different types of level of planning and scheduling. Hermawan is one of them and the authors need to make a complete review in this regard (i.e., see documents provided by PMI, AACE, CII, etc.)\n3) Methods:\nHow did the authors select the experts? Are there any criteria other than being a member of PAPPI? How were the statements gathered? There could be many more statements to be included in the survey? The authors need to support the idea of why these statements have been selected.\n4) Conclusions:\nThis reviewer believes the findings of this research do not make enough contribution to the literature, since these findings have been mentioned in many publications before. Many practitioners know that S-Curve bar charts have many limitations while delay analysis is performed. This is not a new finding.\nOverall, this reviewer believes that this study cannot make enough contribution to the literature.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "6598", "date": "05 May 2021", "name": "Putri Luthan", "role": "Author Response", "response": "To: S. M. Reza Alavipour, Dept. of Civil and Architectural Engineering, Illinois Institute of Technology, Chicago, IL, USA My revision is as follows: Q: 1) Title: The title is very long and the contribution and originality of the research could not be understood by reading the current title. A: The title is the result of discussions between the author and the F1000 Research Publisher. Old title: Comparative Study on the Use of S Curve-Bar Chart and Ms. Project-PDM Methods in Scheduling and Controlling Building Construction Project. New Title: Use of the bar chart/S-curve and computerized precedence diagram method on scheduling and controlling building construction projects by contractors: a cross-sectional study Q: 2) Introduction: There are many more research studies that proposed different types of level of planning and scheduling. Hermawan is one of them and the authors need to make a complete review in this regard (i.e., see documents provided by PMI, AACE, CII, etc.) A: PDM can show the position of project delays in terms of the number of days, then to determine the final cost of the project it can be integrated through the earned value management (EVM) method through the Ms. Project application. The critical trajectory will be a concern from the author as an indicator of the project that is projected to be late. This is what the writer will develop to make a dashboard with Ms. Project as a control tool that is easy to understand. Q: 3) Methods: How did the authors select the experts? Are there any criteria other than being a member of PAPPI? How were the statements gathered? There could be many more statements to be included in the survey? The authors need to support the idea of why these statements have been selected. A: Apart from members of PAPPI, other criteria which qualify as a research sample are: 1. Workers who understand the use of the S-curve and PDM in a construction project 2. Workers in charge of contractors who apply the PDM method to project planning and scheduling Statements on the instrument are compiled based on theoretical studies (content validity) and experience working as planners and field supervisors for building construction projects. Q: 4) Conclusions: This reviewer believes the findings of this research do not make enough contribution to the literature, since these findings have been mentioned in many publications before. Many practitioners know that S-Curve bar charts have many limitations while delay analysis is performed. This is not a new finding. A: This finding contributes to the science of construction management because these empirical findings confirm that large-scale projects should use planning and scheduling using the PDM-MS Project method by paying attention to the critical trajectory and free float of a job. Best Regards Putri Lynna A. Luthan" } ] }, { "id": "89537", "date": "09 Aug 2021", "name": "Anton Soekiman", "expertise": [ "Reviewer Expertise - Construction Engineering and Management" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n1. Title:\nThe title did not explain what is the contribution of the research and could not be understood by reading the current title.\n\n2. Introduction:\nThere are many different types of planning and scheduling methods that propose different levels of planning and scheduling with their respective advantages and disadvantages. However, its application must be adapted to the conditions and situations in the field and the level of human resources involved.\n\n3. Methods:\nOf the two planning and scheduling methods compared, each has its own advantages and disadvantages. However, the questions posed to the respondents did not elaborate on the advantages and disadvantages of the research method.\n\nThe human resources involved in the project vary in their educational levels from top to bottom. The successful application of project planning and scheduling methods needs to take into account the diversity of these levels, so that the respondents of this study should accommodate the various levels of labor involved in the project.\n\nThere are errors in the inclusion of respondents' results in Figures 3, 4, 5 which look the same.\n\n4. Conclusions:\nThe conclusion of this research did not give any new findings so this research does not make enough contribution to the knowledge and practitioners, since these findings are familiar standard opinion and have been mentioned in many publications before.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-284
https://f1000research.com/articles/9-630/v1	19 Jun 20	{ "type": "Research Article", "title": "Cigarette consumption in adult dual users of cigarettes and e-cigarettes: a review of the evidence, including new results from the PATH study", "authors": [ "Peter N. Lee", "John S. Fry", "Barbara A. Forey", "Katharine J. Coombs", "Alison J. Thornton", "John S. Fry", "Barbara A. Forey", "Katharine J. Coombs", "Alison J. Thornton" ], "abstract": "Background: Modelling disease risk from e-cigarette use requires knowing how much e-cigarette uptake affects a smoker’s cigarette consumption. From Waves 1-3 of the US PATH study and other evidence, we sought answers to three questions: (Q1) Does consumption differ between dual users and exclusive smokers? (Q2) Does initiating e-cigarettes affect cigarette consumption? (Q3) Is baseline consumption related to later initiation of e-cigarettes? Methods: Consumption data from PATH were corrected for clear recording errors, with e-cigarette use classified as established or every day, and adjustment made for demographics, use of alcohol, drugs and other tobacco products, and age of starting smoking. Searches identified other studies published since 2008, with Q1 answered from cross-sectional studies, and Q2 and Q3 also from prospective studies. Results: (Q1) In PATH, consumption in current every day e-cigarette users was 2-3 cigarettes per day lower in dual users, with no difference seen in established users. From 31 published studies, consumption was no lower in dual users for current or ever e-cigarette users. (Q2) In PATH study consistent decreases in consumption occurred in those becoming every day dual users with increases seen in those reverting to exclusive smoking. Thirteen published studies consistently showed reduced consumption in those becoming dual users. (Q3) Three studies consistently showed greater consumption in smokers subsequently initiating e-cigarettes, but no significant difference was seen for PATH. Conclusions: Assessment is complicated by the few every day e-cigarette users in PATH, and between-study variability in quantifying e-cigarette use and presentation of results. As taking up e-cigarettes is associated with reducing consumption, finding no clear cross-sectional difference between dual users and exclusive smokers suggests smokers taking up e-cigarettes had higher consumption initially, limited evidence supporting this. Given the much lower disease risk of e-cigarettes vs. cigarettes, smokers becoming dual users should somewhat reduce their disease risk.", "keywords": [ "Smoking", "E-cigarettes", "Dual use", "Cigarette consumption" ], "content": "Abbreviations\n\nCC, cigarette consumption; CI, confidence interval; OR, odds ratio; PATH, Population Assessment of Tobacco and Health; RRTP, reduced-risk tobacco product; SE, standard error.\n\n\nIntroduction\n\nVarious population health impact models have been developed to estimate the extent to which mortality is reduced following the introduction of a reduced-risk tobacco product (RRTP). In many applications of one approach (Lee et al., 2017) it is assumed that adult dual users of cigarettes and RRTPs have a mean cigarette consumption (CC) per day that is half that of cigarette smokers who do not use RRTPs. A substantial reduction in CC in dual users is also assumed in another similar modelling exercise (Poland & Teischinger, 2017). However, other approaches (e.g. (Bachand et al., 2018; Hill & Camacho, 2017; Vugrin et al., 2015)) have assumed that dual users and cigarette-only smokers have the same or a very similar CC. While conclusions regarding CC in dual users may depend on the specific type of RRTP, there is very little evidence available on very recently introduced types, such as heated tobacco products. However, as we demonstrate, a considerable number of papers have published evidence relating to e-cigarettes.\n\nThe objective of the work described here is to review and summarize evidence in adults comparing CC by dual users of cigarettes and e-cigarettes with CC by cigarette-only smokers. More specifically, this study attempts to answer three interrelated questions.\n\nQ1. Does CC differ between dual users and exclusive smokers?\n\nQ2. Does take-up of e-cigarettes change CC?\n\nQ3. Does baseline CC differ by subsequent take-up of e-cigarettes?\n\nAnswers to Q1 may be obtained from cross-sectional studies carried out at one point in time, or from prospective studies at separate follow-ups. Answers to Q2 and Q3 will usually require information from prospective studies, though cross-sectional studies asking questions on the history of tobacco use might also provide relevant results.\n\nWe derive answers to the three questions from Waves 1, 2 and 3 of the publicly available adult data from the Population Assessment of Tobacco and Health (PATH) study (Hyland et al., 2017), a longitudinal cohort study in the U.S. supported by Federal funds, some previous publications (Berry et al., 2019; Buu et al., 2018) having only considered data from Waves 1 and 2. We also derive results from a detailed review of other published literature. We restrict attention to adults as they are most relevant to the estimation of the impact on mortality of RRTP introduction, and as the level of CC in cigarette smoking youths is less firmly established.\n\n\nMethods\n\nTo be included in the analyses, individuals had to satisfy four criteria:\n\n1. Be aged 18 to 54 years at Wave 1 (as e-cigarette use proved to be very uncommon in those aged 55+ years).\n\n2. Be current every day cigarette smokers at each Wave with data available on CC.\n\n3. Have data available on current and former established e-cigarette use at each Wave.\n\n4. Have data available at Wave 1 on nine variables considered as potential predictors of CC (referred to subsequently simply as “predictors” – sex (male, female), age range (18–24, 25–34, 35–44, 45–54 years), highest grade of schooling (less than High School, GED, High School graduate, some college (no degree) or associates degree, bachelor’s degree, advanced degree), Hispanic origin (yes, no), race (white alone, black alone, other), age range when first started smoking cigarettes every day (<18, 18–24, 25–34, 35–44, 45–54, 55+ years), ever used alcohol (yes, no) ever used cocaine or crack (yes, no), ever use of other tobacco products (yes, no) – based on separate variables for traditional cigars, cigarillos, filtered cigars, pipes, hookah, smokeless tobacco, snus and dissolvable tobacco.\n\nIn the PATH dataset, the data on CC were recorded by two variables, a number and a unit (cigarettes per day or packs per day), the intention being to derive daily consumption directly from the number variable if the unit was cigarettes per day and by multiplying the number by 20 if the unit was packs per day. However, for some individuals the consumption data recorded in this way was highly implausible. For example, one individual had a recorded CC of 15 packs per day at Wave 1, 10 cigarettes per day at Wave 2 and 15 cigarettes per day at Wave 3. Here the implied consumption of 300 cigarettes per day is highly implausible and it seems much more likely that the Wave 1 consumption was actually 15 cigarettes per day. Accordingly, as described more fully in Extended data, Additional File 1 (Lee, 2020), a detailed investigation of the CC data at each Wave was carried out, and corrections were made for 84 individuals. There were 75 corrections relating to individuals where the consumption at one Wave was seven or more times the average consumption at the other two Waves. However, there were nine individuals where corrections were applied for other reasons, as described in the Additional File (see Extended data; Lee, 2020). All the analyses reported are based on the corrected CC data.\n\nAnalyses were carried out relating to current and ever established e-cigarette use, based on data for a current established e-cigarette user (one who has ever used, has used fairly regularly, and uses every day or some days) and a former established e-cigarette user (one who has ever used, has used fairly regularly, and currently does not use at all), with ever users being those who were either current or former users. Analyses were also carried out relating to current every day e-cigarette use. Note that for Wave 3, the data used were for e-product use (which also includes e-cigars, e-pipes and e-hookahs) rather than e-cigarette use, data for e-cigarette use not being available for this Wave.\n\nAll the analyses were weighted, based on the weighting factors for Wave 1, and used weighted linear regression analyses. Analyses were carried out with no adjustment for other variables and with adjustment for the other nine predictor variables selected, using a forward stepwise approach, including variables significant at p<0.05.\n\nTo answer Q1, the corrected CC was compared in exclusive cigarette smokers and in dual users, separately at each Wave.\n\nTo answer Q2 (and to provide some additional information), the change in corrected CC between two Waves was compared in four groups – exclusive cigarette smokers at both Waves, dual users at both Waves, dual users only at the second Wave, and dual users only at the first Wave. Separate analyses were carried out for each pair of Waves.\n\nTo answer Q3, the corrected CC at the first of two Waves in those who were then exclusive cigarette smokers was compared in those who did or did not become dual users. Separate analyses were carried out for each pair of Waves.\n\nRelevant data were transferred for analysis to a ROELEE database, and analysed using the ROELEE program (Release 59, Build 49). All these analyses could be run using the R-program (https:/www.r-project.org), using the \"lm\" function including the \"weights=\" option for weighted linear regression, and for stepwise regression using the \"step\" function specifying \"method=forward\" and test=\"F\".\n\nThe search was limited to publications from 2008 onwards, e-cigarette usage not being widely established before then (Barrington-Trimis et al., 2015; Bauld et al., 2014; Bell & Keane, 2014). Potential publications were initially obtained from a search on PubMed on 22 October 2018:\n\n(e-cig[tiab] OR e cig[tiab] or electronic cig[tiab] OR electronic nicotine [tiab] OR \"vaping\"[MeSH Terms] OR \"Electronic nicotine delivery systems\"[MeSH Terms]) AND (Epidemiologic studies[MeSH Terms] OR Epidemiology OR Surveys and questionnaires[MeSH Terms] OR survey[TIAB] )\n\nSubsequently, on 26th July 2019, the search was repeated, using the same keywords as before, but restricted to papers published since 1st January 2018.\n\nAn initial trial was undertaken on the first 100 papers for which free copies were available via PubMed, with PNL screening using the abstract only, and BAF independently screening using the full paper. Based on the high level of agreement between the two methods, it was decided that initial screening on the abstract was satisfactory, but that where there was any doubt the papers would need to be obtained and examined in full. In particular, any paper apparently reporting an epidemiological survey in a general adult population which had enquired about both cigarettes and e-cigarettes should be obtained, even if there was no mention in the abstract of dual use or of the number of cigarettes smoked.\n\nAll papers were then screened by BAF or AJT, with the full paper obtained wherever necessary. Papers were accepted as having data, or as being relevant reviews. No restriction was made as to the type of device referred to by the original papers, whether e-cigarettes, ENDS, ANDS, cigalike or other terminology, or whether containing nicotine or not, except that results specifically referring to heated tobacco products were excluded.\n\nFor each paper, rejected papers were marked in two ways. First, whether they were rejected on the basis of the full paper, abstract or title, this final option only being used where no abstract was available through PubMed and the title and publication type clearly indicated it was not relevant. Papers which were at all debatable or borderline for acceptance were also examined by KJC and PNL before being finally rejected.\n\nSecond, the reason for rejection was coded as follows:\n\n1. Not a study asking individuals about their smoking and e-cigarette usage\n\n2. Study of adolescents, young adults (age 25 or under), or students\n\n3. Study in persons with a pre-existing medical condition\n\n4. Study in persons likely to have unusual smoking and/or e-cigarette habits (e.g. studies of illicit drug users, prisoners, homeless persons, e-cigarette retail employees, medical personnel)\n\n5. Trial of smoking cessation methods or of switching to e-cigarettes\n\n6. Study with <100 participants\n\n7. Study restricted to e-cigarette users (or dual users), or inappropriately restricted on smoking habits or quitting status/intention\n\n8. No results presented distinguishing exclusive cigarette smokers and dual users\n\n9. No results on CC\n\n10. Results on CC not in a useable format (e.g. studies which reported amount smoked overall, but not by exclusive/dual use).\n\n11. Report on the PATH study.\n\nOnly one reason was recorded per paper, usually (but not necessarily) the first from the list above. A note on the reason for rejection was also usually recorded.\n\nThe papers identified as review papers from the PubMed search were examined, looking for references to papers apparently describing an epidemiological survey which had enquired about both cigarettes and e-cigarettes. Except where either the context of the review paper or the title of the secondary paper indicated that the survey was in an ineligible population (corresponding to rejection codes 2–7), new papers were added to the list of candidate papers and at least the abstract was examined. As previously, unless the abstract made clear that the study population was ineligible, the full paper was obtained, regardless of whether dual use or amount smoked were mentioned in the abstract. Further review papers cited in the original set of reviews were also considered, but only if the context in the original review indicated that they referred to amount smoked.\n\nFor each publication, information was collected on the study location, title and type (prospective or cross-sectional), the period when the study was conducted, the age of the population considered, the number of smokers, the definition of e-cigarette use (and whether it contained nicotine) and which of the three questions it could provide answers to. For Q1, the data extracted (separately for current and ever e-cigarette use) included the numbers of dual users of cigarettes and e-cigarettes, and of exclusive cigarette smokers, the CC for each group and the difference, with its statistical significance, where possible. Where distributions were given by grouped amount smoked, the estimates of CC were derived using assumed midpoints. For studies where differences could be estimated, comparisons were made, again separately for current and ever e-cigarette use, of the number showing positive and negative differences, with unweighted means and standard errors estimated overall, and separately for US and European studies. For Q2, data were extracted for studies comparing changes in CC in those becoming or not becoming current e-cigarette users, with the number showing significant differences in each direction summarized. Data were also extracted for studies reporting changes in CC only in those becoming current dual users, with the numbers showing reductions or increases summarized. For Q3, evidence was summarised comparing CC in exclusive cigarette smokers according to whether they later used e-cigarettes, there being too few studies to allow more than a description of the results.\n\n\nResults\n\nA total of 3,543 adults were eligible for the analysis. Table 1 summarizes the results relating to Q1. They show no significant tendency for CC to be associated with current or ever established e-cigarette use, with six of the differences being positive and six negative. In contrast, though based on much smaller numbers of dual users, current every day e-cigarette use is associated with a consistently lower CC, with five of the six estimates significant at p<0.05, and the estimated difference ranging from 1.50 to 2.92 cigarettes per day.\n\na Mean cigarette consumption (CC) for all 3,543 selected individuals.\n\nb The number of those with no e-cigarette use is always equal to 3,543 minus the number of dual users.\n\nc Differences in cigarette consumption are always between dual users and those with no e-cigarette use, according to the definition of e-cigarette use. Significance codes used are + p<0.05 ++ p<0.01 +++ p<0.001 NS p≤0.05.\n\nd In Waves 1 and 3 the differences in cigarette consumption are adjusted for sex, age range, school grade, race, Hispanic origin, ever used other tobacco products, and age at starting to smoke. In Wave 2 the differences are adjusted for the same list of factors except ever used other tobacco products. Significance codes are as for unadjusted differences.\n\nTable 2 summarizes the results relating to changes in e-cigarette status between Waves, with those who are exclusive cigarette smokers at each Wave (“No No”) used as the base for comparisons. The analyses comparing the “No Yes” with the “No No” group are directly relevant to Q2 as defined, while those comparing “Yes No” with “No No” provide information on the reverse association.\n\nThe results relating to established e-cigarette use, with one marginal exception, show no significant tendency for changes in CC to be associated with changes in e-cigarette use. The results for every day e-cigarette use show more evidence of a relationship. While they show no consistent tendency for changes to differ between those who are exclusive cigarette smokers at each Wave (“No No”), and those who are dual users at each Wave (“Yes Yes”), there is a consistent decrease in CC in those who became dual users at the second Wave (“No Yes”), and a consistent increase in those who switch from dual use to exclusive smoking (“Yes No”). While none of the decreases are significant (at p<0.05), four of the increases are highly significant (p<0.001). However, the numbers becoming or ceasing to be dual users are small.\n\na The overall mean changes in cigarette consumption were −0.61 (95% CI −0.85 to −0.38) for Wave 2 minus Wave 1, −0.56 (95% CI −0.81 to −0.32) for Wave 3 minus Wave 1, and 0.05 (95% CI −0.15 to 0.26) for Wave 3 minus Wave 2\n\nb Significance codes used for the differences are + p<0.05 ++ p<0.01 +++ p<0.001 NS p≤0.05\n\nc Adjusted for sex, age range, school grade, Hispanic origin and ever use of other tobacco products\n\nd Adjusted for race\n\ne Adjusted for age range\n\nf Adjusted for sex, school grade and Hispanic origin\n\nTable 3 summarizes the results relating to Q3. The results do not show any consistent difference in baseline CC in those who were not e-cigarette users at baseline according to their subsequent e-cigarette use, whether based on current or ever established or current every day use.\n\na SE – standard error. None of the differences were statistically significant at p<0.05\n\nbAdjusted for sex, age range, school grade, race, Hispanic origin, age of starting to smoke and ever use of other tobacco products\n\ncAdjusted for sex, age range, school grade, race, Hispanic origin and age of starting to smoke cigarettes\n\n\nOther published results\n\nOur original PubMed search identified 1,403 publications, a number which extended to 1,434 from examination of reference lists in review papers and secondary references. From these 55 papers were identified as relevant, which described results from 45 apparently relevant studies. The later search identified 697 publications, of which 342 had already been identified by the first search. Nine of the remaining 355 were selected as relevant, with eight describing new studies and one providing additional results from a study identified earlier.\n\nFurther examination of the papers led to seven of the 53 studies being rejected: the Smoking Toolkit Survey (Beard et al., 2018) a time-series analysis linking prevalence of e-cigarette use with average CC in England; a paper (Morean et al., 2018) describing results from four studies, with the data for dual use and for cigarette use coming from two different surveys (and the other two not concerning tobacco); analyses of multiple internet and mail surveys (Etter & Eissenberg, 2015) where the surveys of e-cigarette users and of smokers were conducted at different times and in different national populations; the Mexico ITC survey (Lozano et al., 2019) as its analysis relating to Q2 was not restricted to those not using e-cigarettes at baseline; the Korea KCHS study (Han, 2019) which only related smoking to use of e-cigarettes due to a price increase; a US GFK study (Weaver et al., 2018) which did not relate CC to e-cigarette use at the same time point; and the California CSC study (Al Delaimy et al., 2015) which only compared smokers who either reported they have used e-cigarettes or will never use e-cigarettes at both surveys.\n\nTable 4 summarizes the characteristics of the 46 studies considered, in order of country within continent. Of the 46 studies, 22 were conducted in the US, five in multiple countries, three in the United Kingdom, three in Italy, two in France and one in each of 11 other countries. Of these, 16 studies involved an element of follow-up, the remaining 30 being of cross-sectional design, with three of these involving multiple cross-sectional studies. The studies mainly covered the whole adult population though some were restricted to younger adults. The number of smokers included varied considerably between studies with 10 involving more than 4,000 and seven less than 250. As will become evident, the studies varied in how e-cigarette use was defined and the questions they could answer. All the papers described the product as e-cigarettes, and it has been assumed that all the products contained nicotine, no study stating otherwise, though many did not give details.\n\na CS = cross-sectional, MCS = multiple cross-sectional, P = prospective\n\nb M = mean\n\nc C = current, C (PY) = current (past year), E = ever, ER = ever regular, R = regular\n\nd Q1 = Does CC differ between dual users and exclusive smokers?\n\nQ2 = Does take-up of e-cigarettes affect time changes in CC?\n\nQ3 = Does baseline CC differ by subsequent take-up of e-cigarettes?\n\ne Not given but estimated from results for one survey\n\nf Not given but around 2,000 households are interviewed per month\n\ng 1,820 were interviewed in 2008, with 1,802 in 2010, 1,530 in 2013 and 1,550 in 2014. Note that in this cohort study, respondents are surveyed each year, with drop-outs replenished by inviting new smokers\n\nh Date not stated. 2017 assumes the study was completed the year before publication\n\ni 46% were aged under 21\n\nj The respondents were stated to be “Mainly from the United States” with no further details given\n\nk Number of smokers not stated, the total participants numbered 937\n\nl 34% of respondents from US, 24% France, 8% UK, 6% Switzerland, 28% other countries\n\nTable 4 also gives references for the publications considered in our analyses. For some studies there were additional publications which provided no useful extra results to those cited: Study 2 (Laverty et al., 2018), study 5 (Andler et al., 2015; INPES, 2014), study 9 (Office for National Statistics, 2016), study 10 (Nelson et al., 2015), and study 27 (Shi et al., 2015).\n\nTable 5 summarizes the results from the 39 studies that presented evidence comparing CC in dual users with that in exclusive cigarette smokers. Of these studies, the evidence related to current e-cigarette use in 25, to ever e-cigarette use in eight and to both current and ever use in six. In some studies, results were presented in terms of the distribution of the subjects by broad categories of cigarette consumption and (as described in the footnotes to Table 5) the overall mean CC could only be estimated by making assumptions about the mean CC for each category.\n\na +, - p<0.05; ++, -- p<0.01; +++, --- p<0.001 NS p≥0.05\n\nWhere a code is not given, the significance level was not reported\n\nb Estimated assuming midpoints of 5, 15 and 30 cigs/day for 0-10, 11-20 and 21+ cigs/day\n\nc Estimated assuming midpoints of 5, 15, 25 and 40 cigs/day for 0-10, 11-20, 21-30 and 31 cigs/day\n\nd Estimated from separate data for current, former and never e-cigarette users\n\ne Estimated from adjusted ORs for forced entry analysis assuming midpoints of 5, 13, 18 and 30 cigs/day for 10 or fewer, 11-15, 16-20 and more than 20\n\nf Estimated assuming midpoints of 2.5, 10 and 25 cigs/day for <5, 5-14 and 15+ cigs/day\n\ng Estimated from adjusted ORs assuming midpoints of 2 and 15 cigs/day for <5 and 5+ cigs/day\n\nh Estimated assuming midpoints of 10 and 30 cigs/day for <1, 1+ packs/day\n\ni Estimated assuming midpoints of 5.5 and 21 cigs/day for <11, 11+ cigs/day\n\nj Results relate to use in the last 30 days\n\nk Estimated assuming midpoints of 2.5, 10, 20 and 35 cigs/day for 1-4, 5-14, 15-24 and 25+ cigs/day\n\nl Median consumption\n\nm E-cigarette users were those using them to quit cigarettes\n\nn Estimated from figure combining results for cigs + ecigs and cigs + cigars + ecigs into dual users\n\no Estimating assuming midpoints of 2.5, 8, 15 and 30 cigs/day for <5, 6-10, 11-20 and 21+ cigs/day\n\np Estimating assuming midpoints of 5 and 20 cigs/day for 10 or fewer and >10 cigs/day\n\nq Estimating assuming midpoints of 8 and 25 cigs/day for 1-15 and >15 cigs/day\n\nr Estimating assuming midpoints of 5 and 20 cigs/day for light (<10) and heavy (>10) cigs/day\n\nOf the 30 studies providing estimates relating to current e-cigarette use, two studies (18 and 22) merely stated there was a lack of association between e-cigarette use and amount smoked. In the other studies, estimates of the difference in CC between dual users and exclusive e-cigarette use could be derived, with two being from study 38 at two different time points. The evidence was conflicting, with 11 estimates showing a lower CC in dual users and 18 a higher CC. Omitting the estimate from study 29 (Pearson et al., 2015) (as it only related to e-cigarette use as a cessation aid) and the earlier estimate from study 37 (Grana et al., 2014) to avoid double-counting, the unweighted mean difference was estimated as +0.03 cigarettes per day reduction (Standard error [SE] 0.36). While there was some evidence of a reduction in the 11 estimates from Europe (1.04) cigarettes per day reduction, standard error [SE] 0.72), the 14 estimates from the USA did not confirm this (0.56 increase, SE 0.29).\n\nThe 14 studies providing estimates relating to ever e-cigarette use were equally inconsistent, with four of the ten estimated differences showing a reduction, and six an increase.\n\nOur searches revealed 17 studies that presented evidence on whether cigarette-only smokers who become dual users change their CC. The top half of Table 6 summarizes results for five studies, all of prospective design, where dual use refers to current use and where changes are compared in those who remain exclusive cigarette smokers (“No No”) and those who become dual users (“No Yes”). All these studies present evidence that reductions in CC are significantly greater in dual users. The bottom half of Table 6 also presented evidence from a further eight studies (one prospective and seven cross-sectional), where changes were only recorded in those who became dual users. Of these, seven showed large reductions, which seem likely to be substantially greater than in those remaining exclusive cigarette smokers, although lack of the relevant data prevents this from being tested, while the other (study 41 (Seto et al., 2016)) showed a smaller reduction. The evidence from these 13 studies clearly indicates that becoming a current e-cigarette user leads to a reduction in CC.\n\na Adjusted for education, income, occupational status, socio-professional category and size of urban unit\n\nb Adjusted for age, sex, education, follow-up NRT use and baseline strengths of urge to smoke\n\nc Results based on 24-month follow-up. Some earlier results are also available for 12-month follow-up (Manzoli et al., 2015)\n\nd Numbers include those who quit during follow-up\n\ne Estimated from data provided\n\nf The 82% who reduced had a mean reduction of 8.9 cigarettes per day, so, assuming the remaining 18% had no reduction, the mean reduction would be 7.3 cigarettes per day\n\ng The terms “drastically” and “slightly” were undefined\n\nh The terms “substantially” and “slightly” were undefined\n\nThe other four studies provided less useful results. Study 22 (Choi & Forster, 2014a) reported that changes in CC were almost identical between those who had never used e-cigarettes and those who had used e-cigarettes for at least one day in the past 30 days at baseline, after adjustment for demographics and baseline CC. These results neither relate to a proper definition of current e-cigarette use, nor to taking up e-cigarettes between baseline and follow-up. Study 27 (Shi et al., 2016) concluded that “among early adopters, ever-use of first generation e-cigarettes to aid quitting cigarette smoking was not associated with improved cessation or with reduced CC, even among heavier smokers” but the basis for this conclusion is most unclear, with no formal statistical analysis described and it being unclear when the e-cigarette use occurred, and whether those who quit during follow-up were excluded. Study 37 (Grana et al., 2014) reported that “e-cigarette use at baseline was not associated with a change in cigarette consumption, controlled for baseline cigarette consumption”. In study 43 (Wu et al., 2015), no difference was seen between exclusive cigarette smokers and dual users at six months follow-up in individuals who continued to smoke. These analyses related to ever rather than current use of e-cigarettes.\n\nIt can also be noted that, as studies restricted to dual users were excluded (see Literature searches, rejection reason 7), the results shown in the bottom half of Table 6 may not be comprehensive.\n\nOur searches revealed only three published studies that presented evidence relevant to whether CC in exclusive cigarette smokers differed according to whether they later used e-cigarettes. Study 5 (Andler et al., 2016), a cross-sectional study which involved 4,752 current smokers of whom 759 also currently used e-cigarettes, noted that, before they started to vape, these individuals smoked 21 cigarettes per day as compared to 11.3 in those who continued to smoke. Though the results presented did not allow precise estimation of the difference in CC between smokers according to whether they later used e-cigarettes, the results clearly suggested that heavier smokers were more likely to start vaping. Study 35 (English et al., 2018), a cross-sectional study involving 245 current smokers, of whom 103 also currently used e-cigarettes, reported that, for each additional cigarette smoked per day prior to using e-cigarettes, individuals were 4.0% (p = 0.001) more likely to use e-cigarettes. Study 45 (Chan et al., 2019), a prospective study, presented results from a logistic regression predicting uptake of vaping in 3,797 smokers. After adjustment for a range of smoking-related and demographic variables, they reported an increasing likelihood of uptake with increasing cigarette consumption, with ORs of 1.13 (95% 0.95-1.35), 1.41 (1.12-1.78) and 1.69 (1.19-2.39) for smokers of 11-20, 21-30 and 31+ cigarettes per day, as compared to 0-10 cigarettes per day.\n\n\nDiscussion\n\nBased on the analyses of the PATH study, there was no evidence from the analysis of Q1 (Table 1) that cigarette smokers who were also current or ever established e-cigarette users had a lower CC than exclusive cigarette smokers. However, cigarette smokers who were also current every day e-cigarette users did have a lower CC by about two to three cigarettes per day. This is consistent with CC in dual users being more likely to be affected in adults who use e-cigarettes more often, occasional e-cigarette use being less likely to affect cigarette smoking habits. These results, based on cross-sectional analysis, are less relevant to the effect that e-cigarette use might have on CC than those based on changes over time (Q2). Here again the results for every day e-cigarette use (Table 2) showed more evidence of an effect, with those becoming dual users consistently decreasing CC and those switching from dual use to exclusive smoking consistently increasing their CC, although only the second result was statistically significant. It is possible that those who decided to take up e-cigarettes might have had a different baseline CC than those who continued to smoke cigarettes exclusively (Q3), but our analyses (Table 3) showed no evidence of this.\n\nTwo limitations of our analyses should be noted. One is the small number of cigarette smokers who were also current every day e-cigarette users, ranging from about 50 to 70 in the relevant analyses. The other is the weakness of the CC data itself. While we have made our best attempt to correct obvious errors in the data, some doubts must remain about the validity of our correction.\n\nTwo previous publications (Berry et al., 2019; Buu et al., 2018) have presented results from the PATH study using data from Waves 1 and 2, neither making any mention of correcting the clearly erroneous CC data. Buu et al. (2018) restricted attention to those who, at Wave 1, had in the past 12 months smoked cigarettes and not used e-cigarettes, and who at Waves 1 and 2 had not used tobacco products other than cigarettes or e-cigarettes. Comparisons were made between those who had or had not used e-cigarettes some day or every day in the past 30 days. The authors reported that a higher frequency of e-cigarette use was associated with a lower CC after controlling for baseline CC and other confounding variables. This finding, though based on different statistical procedures, seems consistent with our finding for Q2.\n\nBased on current cigarette smokers aged 25+ years who were not current e-cigarette users at Wave 1, Berry et al. (2019) presented odds ratios (ORs) and 95% confidence intervals (CIs), adjusted for 12 potential confounding variables, relating at least a 50% reduction in CC between Waves 1 and 2 to new e-cigarette use at Wave 2. While no material difference was noted for experimental e-cigarette use (OR 1.08, 95% CI 0.78 to 1.48) or for some day e-cigarette use (OR 1.00, 95% CI 0.58 to 1.74), a highly significant (p<0.001) effect was noted for every day e-cigarette use (OR 5.70, 95% CI 3.47 to 9.35). Their analyses defined current smokers as smoking every day or some days, whereas ours restricted attention to every day smokers. Nevertheless, though differently expressed, their analyses support our findings for Q2, with those switching to every day e-cigarette use reducing their cigarette consumption.\n\nAs regards Q1, the other published studies identified by the literature search (see Table 5) provide no clear evidence of a relationship of current or ever e-cigarette use to CC. The lack of consistent association seen in the literature aligns with the findings from the PATH study shown in Table 1 for current and ever established e-cigarette use, but not with the results for current every day use. A problem is that very few of the reviewed studies provided results for current regular e-cigarette use, which may be considered comparable to the results for every day e-cigarette use from the PATH study. Also many of the studies only presented results for grouped cigarette consumption, making it difficult to accurately quantify CC.\n\nThe results from the other published studies regarding Q2 shown in Table 6 clearly demonstrate that becoming a current e-cigarette user is associated with a reduction in CC. While this conclusion is consistent with that from the PATH study, the actual decrease in CC associated with uptake of e-cigarettes cannot be directly compared, as few of the 13 studies actually presented estimates of the mean reduction, with others simply giving the numbers reducing (or increasing) consumption, or presenting odds ratios for the probability of reducing cigarette consumption by 50% or more, and only five allowing the reduction to be tested against a suitable control group of continuing exclusive smokers.\n\nAs noted above, only three of the other published studies provided evidence relating to Q3. While these results are all consistent with those who choose to start using e-cigarettes being heavier smokers than those who do not choose to do so, our analyses of the PATH study data found no clear relationship. Given the limited number of studies, more evidence is clearly needed to answer Q3.\n\nAssessment of the overall evidence is unclear, partly due to the between-study differences in how e-cigarette use is quantified, and how the results are presented. Given the quite clear evidence from the analyses addressing Q2 that those taking up e-cigarettes and becoming dual users do reduce their CC, the failure to find any very clear difference in CC between current dual users and exclusive cigarette smokers from the analyses of Q1 would suggest that smokers who subsequently take up e-cigarettes had somewhat higher CC to start with. While the analyses at Q3 are rather limited they do tend to support this view.\n\nAlthough the overall evidence is somewhat difficult to interpret, it appears that exclusive cigarette smokers who become dual users tend to decrease their CC, especially if they use e-cigarettes regularly. The level of reduction in CC is generally fairly modest, and seemingly inconsistent with the assumption that those becoming dual users replace half their cigarettes with e-cigarettes. However, given the much lower claimed disease risks of e-cigarettes relative to cigarettes (Nutt et al., 2014), it seems quite possible that smokers who become dual users will reduce their disease risk to some extent.\n\nOverall, it can be concluded that there is clear evidence that exclusive cigarette smokers who become dual users of cigarettes and e-cigarettes reduce their daily CC. The reduction seen is quite modest in most studies (e.g. two or three cigarettes per day), though some studies show greater reductions. The fact that cross-sectional analyses show little difference in CC between dual users and exclusive cigarette smokers may be due to those who take up e-cigarettes tending to be somewhat heavier cigarette smokers to start with, though more evidence is needed to confirm this. Obtaining accurate overall estimates is made difficult by the different way that studies present their results. Harmonization in the way data on patterns of use of different tobacco products are collected and analysed across studies is warranted.\n\n\nData availability\n\nPATH study data: National Addiction & HIV Data Archive Program: Population Assessment of Tobacco and Health (PATH) Study [United States] Public-Use Files (ICPSR 36498). https://www.icpsr.umich.edu/icpsrweb/NAHDAP/studies/36498/versions/V9 (United States Department of Health and Human Services (USDHHS)).\n\nThe data are available under the Terms of Use as set out by ICPSR, which can be accessed when users start the process of downloading the data.\n\nOpen Science Framework: Cigarette consumption in adult dual users of cigarettes and e-cigarettes. A review of the evidence, including new results from the PATH study. https://doi.org/10.17605/OSF.IO/QH8ZN (Lee, 2020).\n\nThis project contains the following extended data file:\n\nCC in dual users - additional file (DOCX).\n\nExtended data are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nWe thank Esther Afolalu for assistance in acquiring the data from the PATH study, and Zheng Sponsiello-Wang and Christelle Chrea for providing technical comments at various stages. We also thank Jan Hamling for assistance in running the analyses, and Yvonne Cooper and Diana Morris for typing the various drafts of the paper.\n\n\nReferences\n\nAdkison SE, O'Connor RJ, Bansal-Travers M, et al.: Electronic nicotine delivery systems: international tobacco control four-country survey. Am J Prev Med. 2013; 44(3): 207–15. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShi Y, Pierce J, White M, et al.: E-cigarette use, smoking cessation, and change in smoking intensity in 2010/2011 TUS-CPS Longitudinal Cohort. In: Proceedings from Society for Research on Nicotine and Tobacco Annual Meeting, Feb 25-28, 2015, Philadelphia, PA, USA, 25. 2015.\n\nShi Y, Pierce JP, White M, et al.: E-cigarette use and smoking reduction or cessation in the 2010/2011 TUS-CPS longitudinal cohort. BMC Public Health. 2016; 16(1): 1105. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStein JS, Heckman BW, Pope DA, et al.: Delay discounting and e-cigarette use: An investigation in current, former, and never cigarette smokers. Drug Alcohol Depend. 2018; 191: 165–173. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSung B: E-cigarette use and smoking cessation among South Korean adult smokers: A propensity score-matching approach. Asia Pac J Public Health. 2018; 30(4): 332–341. PubMed Abstract \| Publisher Full Text\n\nTwyman L, Bonevski B, Paul C, et al.: Electronic cigarettes: Awareness, recent use, and attitudes within a sample of socioeconomically disadvantaged Australian smokers. Nicotine Tob Res. 2016; 18(5): 670–7. PubMed Abstract \| Publisher Full Text\n\nUnited States Department of Health and Human Services (USDHHS): Population assessment of tobacco and health (PATH) Study [United States]Public-Use Files (ICPSR 36498-V9). Last Updated: 2019-11-11. National Instutute of Health. National Institute on Drug Abuse, and United States Department of Health and Human Services. Food and Drug Administration. Center for Tobacco Products. Publisher Full Text\n\nVardavas CI, Filippidis FT, Agaku IT: Determinants and prevalence of e-cigarette use throughout the European Union: a secondary analysis of 26 566 youth and adults from 27 Countries. Tob Control. 2015; 24(5): 442–8. PubMed Abstract \| Publisher Full Text\n\nVugrin ED, Rostron BL, Verzi SJ, et al.: Modeling the potential effects of new tobacco products and policies: a dynamic population model for multiple product use and harm. PLoS One. 2015; 10(3): e0121008. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWackowski OA, Bover Manderski MT, Delnevo CD, et al.: Smokers' early e-cigarette experiences, reasons for use, and use intentions. Tob Regul Sci. 2016; 2(2): 133–145. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang JB, Olgin JE, Nah G, et al.: Cigarette and e-cigarette dual use and risk of cardiopulmonary symptoms in the Health eHeart Study. PLoS One. 2018; 13(7): e0198681. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWeaver SR, Huang J, Pechacek TF, et al.: Are electronic nicotine delivery systems helping cigarette smokers quit? Evidence from a prospective cohort study of U.S. adult smokers, 2015-2016. PLoS One. 2018; 13(7): e0198047. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWu QJ, Xiang YB, Yang G, et al.: Vitamin E intake and the lung cancer risk among female nonsmokers: a report from the Shanghai Women's Health Study. Int J Cancer. 2015; 136(3): 610–617. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWu SY, Wang MP, Li WH, et al.: Does electronic cigarette use predict abstinence from conventional cigarettes among smokers in Hong Kong? Int J Environ Res Public Health. 2018; 15(3): 400. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZavala-Arciniega L, Reynales-Shigematsu LM, Lozano P, et al.: Patterns of awareness and use of electronic cigarettes in Mexico, a middle-income country that bans them: Results from a 2016 national survey. Prev Med. 2018; 116: 211–218. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhuang YL, Cummins SE, Sun YJ, et al.: Long-term e-cigarette use and smoking cessation: a longitudinal study with US population. Tob Control. 2016; 25(Suppl 1): i90–i95. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "79330", "date": "16 Mar 2021", "name": "Lai Wei", "expertise": [ "Reviewer Expertise Tobacco use behavior research." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummary: The authors conducted secondary analysis of cross-sectional and longitudinal PATH data to compare cigarette consumptions among dual users of cigarettes and e-cigarettes. The authors also present a review of published literature on this topic. These reviewers has the following comments.\n\nThe authors describe e-cigarettes as “reduced-risk tobacco products (RRP)”. While there is no doubt that e-cigs have considerably lower exposure to many of the HPHCs compared to combustible cigarettes and therefore present lower risks relative to cigarettes, the evidence is still emerging regarding these products. The authors should therefore consider identifying these products as “Potential Reduced-Risk Products” (PRRP).\n\nWhile we understand that recall bias may complicate inferences on tobacco product use history and the “correction” applied by the authors seems reasonable, the authors should consider including a results table for descriptive statistics (with and without correction) in the Supplement, to allow the reader judge for themselves regarding impact of this correction.\n\nThe authors conclude that “smokers who become duals users will reduce their disease risk to some extent”. This is an extremely broad generalization, particularly given the authors own conclusion that the reduction in cigarettes is “fairly modest”. There is no empirical evidence to support this statement of risk reduction among the entire category of dual users, even to “some extent”. A recent report indicates that dual users are a very heterogenous group[ref-1],[ref-2]. Indeed, established every day frequent e-cigarette users may smoke far fewer cigarettes than current smokers thereby substantially reducing exposure to many of the HPHCs. This may lead to lowering of disease risks. In order to achieve the optimum outcome of reducing smoking related diseases, adult smokers must switch completely from cigarettes to e-cigs. We recommend that the authors either delete or modify the statement with appropriate caveats.\n\nThere are several points for consideration to refine the methodology used in this manuscript:\nIn the method section, the authors describe that the analysis is limited to every day cigarette smokers. The authors described “all the analyses were weighted, based on weighting factors for Wave 1”. In PATH user guide (Section 5), there are various types of weights. It is not clear whether the cross-sectional analysis (Q1) was conducted separately using cross-sectional weights of each wave to ensure the representativeness of the results. Longitudinal analysis should be conducted using longitudinal weights. In this case, wave 3 weights should be applied to answer research questions 2 and 3. The authors must clearly specify which weight was used for each analysis (Q1-Q3). The data is limited to ‘current everyday cigarette smokers at each Wave with data available on CC’. As reported by Borland et. al. (2019)[ref-3] and Smith et al. (2021)[ref-1] cigarette consumption differs significantly between every day and some day cigarette smokers and different segment of dual users. To obtain a complete picture of the current cigarette smoker population, the authors should consider including some day smokers in the analysis. This group more likely represents a larger proportion of dual users and provides a more comprehensive view of the cigarette consumption data for dual users. We note that indeed, there are cigarette consumption data in PATH for some day smokers. To answer Q3, the cigarette consumption was “compared in those who did or did not become dual users”. However, since Q3 is asking “is baseline consumption related to later initiation of e-cigarettes”. A logistic regression model with e-cigarette initiation as the outcome measure and cigarette consumption being the covariate would be more appropriate to answer Q3. Based on the current analysis description for Q3, the analysis will compare exclusive cigarette smokers who did or did not become dual users. A clearer description of ‘dual user’ needs to be given as it is shown in Table 3 there are three types of ‘Definition of Use’. Does the heading ‘Became users’ in the ‘Ever Established’ row mean exclusive daily smokers who became ever established users of e-cigarette? Please clarify. The authors should clearly describe if R survey package is used in the analysis. While the R-program is appropriate to conduct statistical analysis, R survey package allows the researcher to utilize the full-sample weight, variance estimation method, BRR-Fay replicate weights, and that Fay’s factor are correctly specified (See PATH User Guide, Appendix B for example R program code). The authors must specify whether replicate weights and Fay’s correction factor were used in current analyses. Authors state that “nine predictor variables [were] selected, using a forward stepwise approach, including variables significant at p<0.05”. It is not clear what variables were considered and the rationale for selection of the variables... The age range category “55+ years” for the variable ‘age range when first started smoking cigarettes every day’ should not be included as the data analysis is limited to 18 to 54 years. Authors only included respondents “aged 18 to 54 years at Wave 1 (as e-cigarette use proved to be very uncommon in those aged 55+ years”. A quick analysis revealed that by excluding individuals aged 55 or older, around 14% of the sample size is excluded among both current established and former established e-cigarette users at Wave 1. This phrase “(as e-cigarette use proved to be very uncommon in those aged 55+ years)” should be deleted since the data does not appear to support the statement.\n\nIn the result section, the table captions should include more detail descriptions and definitions. For all result tables, the phrase ‘among every day smokers’ should be added if the analysis is only conducted among every day smokers. Does the table footnote ‘NS’ mean ‘not significant’? If yes, the corresponding footnote should be ‘p>0.05’ instead of ‘p<=0.05’. The ‘dual use’ term becomes very confusing when it is corresponding to three meanings (i.e., current establish e-cigarette use, ever established e-cigarette use and current every day e-cigarette use). The authors should be more precise.\nTable 1. Table caption should include ‘among every day smokers’ if the analysis is only conducted among every day smokers. There are three columns of ‘dual users’. The author needs to clarify the definitions of these three types of ‘dual users’. Table 2. The word ‘e-cigarette’ should be added to ‘current established use’, ‘ever established use’, and ‘current every day use’. The ‘No No’, ‘Yes Yes’, ‘No Yes’, ‘Yes No’ combinations should be clearly labeled with CS and ECIG to clearly identify the groups being considered. Table 3. The word “e-cigarette” should be specified in the ‘Definition of Use’ column. And the dual use needs to be clearly defined (see previous comments in the method section).\n\nLiterature review results\nResults related to Q2 In the literature review section, some of the findings were drawn from studies with adolescent data. Five out of 13 studies (Table 6) referenced in the results include adolescent data. The authors should be consistent with the literature search criteria and either exclude studies on youth as that was considered out of scope for the review, modify their search criteria or provide a rationale regarding inclusion of only select publications with adolescent data. Results related to Q3. Based on Andler et al. 2016, the authors state that ‘heavier smokers were more likely to start vaping’. However, this does not accurately represent the results from the publication. Andler et al. 2016, state that ”82 % of dual users reported that vaping had enabled them to reduce their cigarette consumption, with a mean reduction of 8.9 cigarettes per day. These individuals were initially heavy smokers, smoking 21 cigarettes per day on average (the mean overall among smokers was 11.3 in 2014.” Just because the participants in the study were heavy smokers that does not establish causality. Furthermore, Andler et al. 2016, included adolescents (age 15+) in their study. The authors should consider providing more context around the statement on heavy smokers.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "6515", "date": "12 Apr 2021", "name": "Peter Lee", "role": "Author Response", "response": "Reply to comments from Drs Wei, Lizhnyak and Sarkar on March 16 2021 We thank the three reviewers for the time they spent and their helpful comments. Our responses are shown in italics below each comment. We hope that the changes we have made to the paper will allow the reviewers’ to approve the revision without reservations. Approved With Reservations Summary: The authors conducted secondary analysis of cross-sectional and longitudinal PATH data to compare cigarette consumptions among dual users of cigarettes and e-cigarettes. The authors also present a review of published literature on this topic. These reviewers had the following comments. The authors describe e-cigarettes as “reduced-risk tobacco products (RRP)”. While there is no doubt that e-cigs have considerably lower exposure to many of the HPHCs compared to combustible cigarettes and therefore present lower risks relative to cigarettes, the evidence is still emerging regarding these products. The authors should therefore consider identifying these products as “Potential Reduced-Risk Products” (PRRP). We actually used the abbreviation RRTP. As the abbreviation is only used in the introduction section in a general context, not specific to e-cigarettes, we think it remains valid. Though we accept that there is currently no good epidemiological evidence relating to e-cigarettes, the statements we make in the paper concerning risk, which are few, are still valid. Thus in the discussion we only state “However, given the much lower claimed disease risks of e-cigarettes relative to cigarettes (Nutt et al., 2014), it seems possible that smokers who become dual users will reduce their disease risk to some extent,”, and in the abstract we say “Given the much lower disease risk of e-cigarettes vs. cigarettes, smokers becoming dual users should somewhat reduce their disease risk.” While we understand that recall bias may complicate inferences on tobacco product use history and the “correction” applied by the authors seems reasonable, the authors should consider including a results table for descriptive statistics (with and without correction) in the Supplement, to allow the reader judge for themselves regarding impact of this correction. As specified in the paper, some of the reported cigarette values are clearly wrong and by a factor of 20. This is not due to recall bias, but to errors by the participant regarding the units used, for example wrongly recording they smoked 20 packs a day, when they meant 20 cigarettes a day. We would certainly not wish to present any results based on the uncorrected values and strongly recommend that no-one else does so. The authors conclude that “smokers who become dual users will reduce their disease risk to some extent”. This is an extremely broad generalization, particularly given the authors own conclusion that the reduction in cigarettes is “fairly modest”. There is no empirical evidence to support this statement of risk reduction among the entire category of dual users, even to “some extent”. A recent report indicates that dual users are a very heterogenous group[ref-1],[ref-2]. Indeed, established every day frequent e-cigarette users may smoke far fewer cigarettes than current smokers thereby substantially reducing exposure to many of the HPHCs. This may lead to lowering of disease risks. In order to achieve the optimum outcome of reducing smoking related diseases, adult smokers must switch completely from cigarettes to e-cigs. We recommend that the authors either delete or modify the statement with appropriate caveats. It is important to note that the phrase cited by the reviewers, from the abstract, “smokers who become dual users will reduce their disease risk to some extent, is preceded by the conditional statement “Given the much lower disease risk of e-cigarettes vs. cigarettes”. This links back to our answer to point 1. Given that e-cigarettes are much less risky than current cigarettes (the generally accepted view) and given that smokers switching to dual use replace some of their cigarettes by e-cigarettes, then smokers who become dual users will indeed reduce their disease risk. The reviewers recommended that we modify the statement with an appropriate caveat, but we had already done so. There are several points for consideration to refine the methodology used in this manuscript: In the methods section, the authors describe that the analysis is limited to every day cigarette smokers. The authors described “all the analyses were weighted, based on weighting factors for Wave 1”. In PATH user guide (Section 5), there are various types of weights. It is not clear whether the cross-sectional analysis (Q1) was conducted separately using cross-sectional weights of each wave to ensure the representativeness of the results. Longitudinal analysis should be conducted using longitudinal weights. In this case, wave 3 weights should be applied to answer research questions 2 and 3. The authors must clearly specify which weight was used for each analysis (Q1-Q3). As the population analysed was restricted to those with relevant data at all three Waves, it seemed appropriate to us to use the Wave 1 person weights throughout both for simplicity and to ensure comparability between all our findings. Clearly, some of the analyses could have used other weights, but we prefer to keep to what we did. The data is limited to ‘current everyday cigarette smokers at each Wave with data available on CC’. As reported by Borland et. al. (2019)[ref-3] and Smith et al. (2021)[ref-1] cigarette consumption differs significantly between every day and some day cigarette smokers and different segment of dual users. To obtain a complete picture of the current cigarette smoker population, the authors should consider including some day smokers in the analysis. This group more likely represents a larger proportion of dual users and provides a more comprehensive view of the cigarette consumption data for dual users. We note that indeed, there are cigarette consumption data in PATH for some day smokers. The problem is that the reporting of cigarette consumption in the PATH study for someday smokers is not comparable to that for everyday smokers. Whereas everyday smokers were asked the average number of cigarettes now smoked per day, someday smokers were only asked about the average number of cigarettes smoked in the past 30 days on the days they were smoking, so we do not know their average daily consumption. While it is possible that some extra information might have been derived using also data for someday smokers (or different weights) we prefer to describe what we did, and let others report alternative analyses they might think are preferable. To answer Q3, the cigarette consumption was “compared in those who did or did not become dual users”. However, since Q3 is asking “is baseline consumption related to later initiation of e-cigarettes”. A logistic regression model with e-cigarette initiation as the outcome measure and cigarette consumption being the covariate would be more appropriate to answer Q3. In response to this point, we ran analyses equivalent to those used to answer Q3, but using weighted logistic regression as suggested by the reviewers rather than weighted linear regression as we had done. The unadjusted results were identical, and the adjusted results gave such similar results that it seems unnecessary to revise our results. Based on the current analysis description for Q3, the analysis will compare exclusive cigarette smokers who did or did not become dual users. A clearer description of ‘dual user’ needs to be given as it is shown in Table 3 there are three types of ‘Definition of Use’. Does the heading ‘Became users’ in the ‘Ever Established’ row mean exclusive daily smokers who became ever established users of e-cigarette? Please clarify. We have amended Table 3 to make it clear (in a footnote) that all the analyses concerned those who were current everyday cigarette smokers at all three waves, and to make it clear that the column “Definition of use” relates to use of e-cigarettes. The heading already makes it clear that we are concerned with baseline non e-cigarette users who became e-cigarette users at follow-up. The authors should clearly describe if R survey package is used in the analysis. While the R-program is appropriate to conduct statistical analysis, R survey package allows the researcher to utilize the full-sample weight, variance estimation method, BRR-Fay replicate weights, and that Fay’s factor are correctly specified (See PATH User Guide, Appendix B for example R program code). The authors must specify whether replicate weights and Fay’s correction factor were used in current analyses. No we have not used this technique. We state in the methods section that “All the analyses were weighted, based on the weighting factors for Wave 1, and used weighted linear regression analyses”. We also state that: “Software used to analyse data from the PATH study. Relevant data were transferred for analysis to a ROELEE database, and analysed using the ROELEE program (Release 59, Build 49). All these analyses could be run using the R-program (https:/www.r-project.org), using the \"lm\" function including the \"weights=\" option for weighted linear regression , and for stepwise regression using the \"step\" function specifying \"method=forward\" and test=\"F\" . Authors state that “nine predictor variables [were] selected, using a forward stepwise approach, including variables significant at p<0.05”. It is not clear what variables were considered and the rationale for selection of the variables... The first paragraph of the methods section, sub-section 4, lists the nine predictor variables (sex, age, education, Hispanic origin, race, age starting cigarettes, alcohol, cocaine/crack, and other tobacco products. These were included into the final model, including the endpoint of interest, using forwards stepwise approach. These candidate variables were selected from a general consideration of the literature and not from stepwise regression from some longer list. The section of the methods says “Analyses were carried out with no adjustment for other variables and with adjustment for the other nine predictor variables selected, using a forward stepwise approach, including variables significant at p<0.05.” We have changed this sentence to read “Analyses were carried out with no adjustment for other variables and with adjustment for those of the nine predictor variables described above which were selected using a forward stepwise approach to include variables significant at p<0.05.” The age range category “55+ years” for the variable ‘age range when first started smoking cigarettes every day’ should not be included as the data analysis is limited to 18 to 54 years. In fact the analyses included those aged 18 to 64 years, as we now make clear in the revised version of the paper. Authors only included respondents “aged 18 to 54 years at Wave 1 (as e-cigarette use proved to be very uncommon in those aged 55+ years”. A quick analysis revealed that by excluding individuals aged 55 or older, around 14% of the sample size is excluded among both current established and former established e-cigarette users at Wave 1. This phrase “(as e-cigarette use proved to be very uncommon in those aged 55+ years)” should be deleted since the data does not appear to support the statement. We have amended the sentence for point 1 of paragraph 1 of the methods to read “Be aged 18 to 64 years at Wave 1, as preliminary analysis showed e-cigarette use to be rare at older ages. Thus, for example, only 9 out of 472 (1.9%) ever established users at Wave 1 were aged 65+ years, and only 18 out of 835 (2.2%) were.” We have also added 55-64 years to the categories of the age range in point 4 of paragraph 1. In the result section, the table captions should include more detail descriptions and definitions. For all result tables, the phrase ‘among every day smokers’ should be added if the analysis is only conducted among every day smokers. Does the table footnote ‘NS’ mean ‘not significant’? If yes, the corresponding footnote should be ‘p>0.05’ instead of ‘p<=0.05’. We have corrected the relevant parts of footnotes in Tables 1 and 2 to read “NS (not significant) p>0.05” The ‘dual use’ term becomes very confusing when it is corresponding to three meanings (i.e., current establish e-cigarette use, ever established e-cigarette use and current every day e-cigarette use). The authors should be more precise. Table 1. Table caption should include ‘among every day smokers’ if the analysis is only conducted among every day smokers. We have amended the footnote of Table 1 to emphasise that the analyses are based on those who were everyday cigarette smokers at all three waves, though this is already stated in the Methods section. There are three columns of ‘dual users’. The author needs to clarify the definitions of these three types of ‘dual users’. We have amended the footnote of Table 1 to make it clearer that dual users are those who are both everyday cigarette smokers and e-cigarette users as defined in the column headings. Table 2. The word ‘e-cigarette’ should be added to ‘current established use’, ‘ever established use’, and ‘current every day use’. We have done this. The ‘No No’, ‘Yes Yes’, ‘No Yes’, ‘Yes No’ combinations should be clearly labeled with CS and ECIG to clearly identify the groups being considered. The labels ‘No No’ etc only apply to e-cigarette use, as all the individuals considered are everyday cigarette smokers at all three waves. This has been made clearer in the Table. Table 3. The word “e-cigarette” should be specified in the ‘Definition of Use’ column. We have done this. And the dual use needs to be clearly defined (see previous comments in the method section). The word “dual” is not used. Each column is labelled by one of the three e-cigarette use categories, and the results are all within everyday cigarette smokers at all three waves, as has been made clear. Literature review results Results related to Q2 In the literature review section, some of the findings were drawn from studies with adolescent data. Five out of 13 studies (Table 6) referenced in the results include adolescent data. The authors should be consistent with the literature search criteria and either exclude studies on youth as that was considered out of scope for the review, modify their search criteria or provide a rationale regarding inclusion of only select publications with adolescent data. In the methods section, we have clarified the second reason for rejection as “Study only of adolescents, young adults (age 25 or under), or students”. We did not want to exclude studies that included adolescents as well as adults. Results related to Q3. Based on Andler et al. 2016, the authors state that ‘heavier smokers were more likely to start vaping’. However, this does not accurately represent the results from the publication. Andler et al. 2016, state that ”82 % of dual users reported that vaping had enabled them to reduce their cigarette consumption, with a mean reduction of 8.9 cigarettes per day. These individuals were initially heavy smokers, smoking 21 cigarettes per day on average (the mean overall among smokers was 11.3 in 2014.” Just because the participants in the study were heavy smokers that does not establish causality. Furthermore, Andler et al. 2016, included adolescents (age 15+) in their study. The authors should consider providing more context around the statement on heavy smokers. We have amended the statement “the results clearly suggested that heavier smokers were more likely to start vaping” to read “the results clearly suggested that vapers were more likely to be heavier smokers." } ] } ]	1	https://f1000research.com/articles/9-630
https://f1000research.com/articles/10-282/v1	12 Apr 21	{ "type": "Research Article", "title": "Health related quality of life in COVID-19 survivors discharged from acute hospitals: results of a short-form 36-item survey", "authors": [ "Alessia Saverino", "Eva Zsirai", "Raphael Sonabend", "Lorenza Gaggero", "Isabella Cevasco", "Caterina Pistarini", "Paolo Cremonesi", "Eva Zsirai", "Raphael Sonabend", "Lorenza Gaggero", "Isabella Cevasco", "Caterina Pistarini", "Paolo Cremonesi" ], "abstract": "Background: Health-related quality of life (HRQL) is important for evaluating the impact of a disease in the longer term across the physical and psychological domains of human functioning. The aim of this study is to evaluate HRQL in COVID-19 survivors in Italy using the short form 36-items questionnaire (SF-36). Methods: This is an observational study involving adults discharged home following a coronavirus disease 2019 (COVID-19)-related hospital admission. Baseline demographic and clinical data including the Cumulative Illness Rating Scale (CIRS) and the Hospital Anxiety and Depression Scale (HADS) were collected. The validated Italian version of SF-36 was administered cross-sectionally. The SF-36 contains eight scales measuring limitations in physical and social functioning, the impact on roles and activities, fatigue, emotional well-being, pain and general health perception. Results: A total of 35 patients, with a mean age of 60 years, completed the SF-36. The results showed difficulties across the physical and psychological domains, particularly affecting the return to previous roles and activities. A higher burden of co-morbidities as well as a more severe muscle weakness was associated to a lower physical functioning. Younger age, rather than older, correlated to a perceived greater limitation in physical functioning and vitality. Conclusions: COVID-19 survivors particularly the ones of working age may need support for resuming their premorbid level of functioning and returning to work.", "keywords": [ "Health Related Quality of Life", "long-COVID", "return to work" ], "content": "Introduction\n\nMore than 100 million cases of coronavirus disease 2019 (COVID-19) have been reported worldwide since January 2020.1 Recent publications have described various persistent physical, cognitive and psychological symptoms in COVID-19 survivors, also named “long-COVID”,2 following their discharge from acute hospital units.3–5 These symptoms include fatigue, difficulty in breathing, difficulty in concentration and memory, pain, anxiety, depression and symptoms of post-traumatic stress disorder. Some authors have found an association of these symptoms to reduced functioning and performance in activities of daily living (ADL)6 with an improvement when a period of rehabilitation was offered.6,7 These findings appear to confirm what was expected based on previous literature on post ITU syndrome8–10 and pulmonary rehabilitation11,12 and have an important implication for implementing follow up services and rehabilitation pathways.13,14\n\nHealth-related quality of life (HRQL) is an important domain for evaluating the impact of a disease in the longer term, which can be measured by a large variety of either disease-specific or generic questionnaires. The Short Form 36 Health Survey (SF-36) is one of the most widely used generic HRQL questionnaires. It was developed in the USA for use in the RAND corporation's health insurance experiment15 and has subsequently been used around the world to gauge the health of populations and to help with service planning. A validated Italian version is available.16 The SF-36 contains eight scales, assessing the quality of life across different domains with physical and mental components. The SF-36 has been found to be a valid instrument to measure HRQL in patients with other chronic respiratory problems such as COPD17 and idiopathic pulmonary fibrosis.17 HRQL has been evaluated in a post COVID-19 population at one month following their discharge from acute hospitals in Wenzhou, China,18 showing significantly poorer SF 36 scores, particularly in the dimensions of physical and emotional role (RP and RE) as well as social functioning (SF), when compared to the Chinese population norm. Based on their findings, these authors highlighted the importance of addressing physical and psychological long-term suffering in post COVID-19 patients.\n\nPreliminary results have been published to evaluate the response to pulmonary rehabilitation in a cohort of post COVID-19 elderly patients,7 showing a positive effect both on respiratory function as well as on HRQL.\n\nThe focus during the acute COVID-19 infection is on the survival of the patient followed by physical functioning in basic ADLs in the post-acute phase.19 In the authors’ opinion other domains beyond basic ADLs, such as role limitations, vitality, and social and emotional functions become at least as important once patients are discharged home expecting to resume their previous roles and functioning.\n\nThe aim of our study is to determine the quality of life in a group of COVID-19 survivors following their discharge from acute hospitals using the SF-36 questionnaire.\n\n\nMethods\n\nThis was a descriptive observational study. A telephone survey was administered cross-sectionally by one of the researchers (IC) to COVID-19 survivors following their discharge from hospital. The researcher administering the survey was unknown to the patients and unrelated to the delivery of their care in order to reduce a potential social desirability bias i.e. patients reporting a more positive outcome to please the treating staff.\n\nThe outcomes of the survey were compared to normative data as well as a range of baseline clinical tests. Being a non- interventional study with an unknown prevalence of the variable under investigation, the sample size was not calculated.\n\nAll patients had an initial admission to an acute hospital unit in Genoa, Italy, including intensive care units (ICUs) and general acute COVID-19 wards, followed by an interim stay in a subacute unit (Nave Ospedale unit, Genoa) before their discharge home. This interim admission was arranged to free beds in acute units and step-down patients to a non-acute level of medical care. Two consecutive negative COVID-19 tests were considered necessary criteria for discharge at that time. Although at this stage patients were encouraged to walk and actively engage in ADLs under the supervision of the nursing staff, they did not receive any specific physiotherapy or pulmonary rehabilitation.\n\nAll patients admitted to the Nave Ospedale unit from the 6th May and discharged within the 4th June 2020, who were able to understand the informed consent protocol and complete the survey in Italian, were considered eligible. Patients were approached within the first week of their admission and written informed consent was taken.\n\nThis study was registered within the Ospedali Galliera Scientific Committee and approved by the Regione Liguria Ethics Committee (CER Liguria, RP/52/UCS). All participants gave written informed consent to participate in the study.\n\nDemographic and clinical characteristics were collected whilst patients were admitted to the Nave unit. This included age, sex, illness burden measured by the Cumulative Illness Rating Scale (CIRS),20 the modified Medical Research Council Dyspnea scale (mMRC-D),21 the 30 second sit to stand test (30sSTS),22 and the Hospital Anxiety and Depression Scale (HADS).23 The mMRC-D describes the level of difficulty in breathing experienced at different levels of activity intensity. The 30sSTS is a standard measure for lower limb muscle strength and measures the number of times a patient can stand up from a chair in 30 seconds. The HADS is a self-reported questionnaire for screening anxiety and depression in community and hospital medical practice, which provides a total anxiety and depression score, where 8-10 is considered borderline abnormal and 11-21 abnormal.\n\nThe SF-36 survey was administered at a single point in time on the 13th June 2020 to the patients who had been discharged from the Nave Ospedale unit in the previous 2 months.\n\nThe SF-36 includes one multi-item scale which is feasible to be administered to individuals over the age of 14 by a telephone interview.24 The survey includes eight scales measuring HRQL across physical and psychological domains: 1) limitations in physical functioning (PF) because of health problems; 2) limitations in usual role activities because of physical health problems (RP); 3) limitations in usual role activities because of emotional problems (RE); 4) energy and fatigue (EF); 5) emotional well-being (EWB); 6) social functioning (SF); 7) bodily pain (BP); and 8) general health perceptions (GH). All questions are scored on a scale of 0 to 100, with 100 representing the highest level of functioning. The scores belonging to each scale are averaged together to give eight total scores.\n\nSummary statistics are presented as mean, standard deviation (SD), and range for continuous variables (age; time between acute admission and SF-36; time from discharge to SF-36; CIRS; mMRC-D; 30sSTS; HADS), and as counts with percentages for categorical variables (sex) (Table 1). Testing for significant differences in discrete variable distributions was carried out with the Chi-Squared (χ2) test. Continuous variables are tested for association with linear regression modelling and associated t-tests, and continuous and discrete variables are compared with the Kolmogorov-Smirnov test. Two-sample difference of means t-tests are initially conducted on the continuous variables from a simple linear regression analysis and then with multiple regression to account for possible confounding of age and sex. Correlations for continuous variables are reported as Spearman’s rank correlations with p-values, Pearson correlations were also calculated for continuous variables in order to support the Spearman correlations, but these are not reported. The SF-36 scale scores are compared to the normative data for the Italian population15 via two-tailed t-tests. Benjamini-Hochberg multiple testing correction was used to adjust for the possibility that significant p-values may occur by chance. Adjusted p-values are reported and a significant result is taken to be one with a (adjusted) p-value less than or equal to 0.05. There were no missing data in the SF-36 outcomes and missing data in other variables was handled by case-wise deletion when required in analysis, sample sizes are reported alongside results. Analysis was performed with R version 4.0.2.\n\n\nResults\n\nBaseline demographic and clinical characteristics of the population are reported in Table 1.27 A total of 45 out of 80 (56%) patients were eligible for the study and agreed to participate, of which 35 patients completed the SF-36 questionnaire. Of these, 12 were female and 23 were male, with a mean age of 60 years (±15) ranging from 30 to 85 years; 63% were below 65 years. The average CIRS score was 9 (±4.3), ranging from 3 to 25.\n\nAt the time of the SF-36 administration, the average time in days since the initial hospital admission for COVID-19 related symptoms was 61.8 days (±15.5), ranging from 20 and 95, while the average time from the discharge from acute hospitals was 39 days (±14.8), ranging from 15 and 71 days.\n\nTable 2 shows the scores for the different SF-36 domains. The mean (SD) SF-36 scales scores in ascending order (from the lowest to the highest) were as follows: limitations in role physical RP = 6.4 (17.5); limitations in role emotional problems RE = 30.5 (27.2); social functioning SF = 48.2(19.2); general health perceptions GH = 42.4 (17.7); energy and fatigue EF = 48.4 (15.3); physical functioning PF = 51.6 (24.4); emotional well-being EWB = 60.2 (16.8); bodily pain BP = 60.8 (19.2). All scores were significantly different from the normative data (p < 0.01) except emotional wellbeing (t = −1.79, p = 0.07).\n\nHigher age was significantly associated with higher physical functioning (t = 4.34, p = 0.01; r(32) = 0.57, p = 0.01) and higher energy fatigue (t = 3.14, p = 0.05; r(32) = 0.49, p = 0.03). Whilst age was significantly associated to higher general health (t = 3.09, p = 0.05), the correlation is non-significant (p = 0.09). Higher performance on the 30sSTS test was significantly associated to higher physical functioning (t = 4.01, p = 0.02; r(23) = 0.63, p = 0.01). Finally higher CIRS was significantly associated with lower physical functioning (t = -3.63, p = 0.03; r(33) = -0.58, p < 0.01), as well as being significantly correlated with poorer general health (r(33) = -0.57, p = 0.01). Time between acute admission and SF-36, as well as time from discharge to SF-36, sex, MRC-D, HADS A and HADS D showed non-significant correlation to SF-36 scores. Correlations for the components of the SF-36 with demographic and clinical factors are shown in Table 3. After adjusting for age, only age remained significantly associated with the SF-36 scores. The full results from univariate and multivariate analysis are provided as supplementary material.\n\nOnly significant values are reported, dashes indicate non-significant.\n\n\nDiscussion\n\nWe describe the impairment of HRQL in both physical and psychological functioning in COVID-19 survivors about a month (average 39 days) following their discharge from an acute hospital unit. All eight scales of the SF-36 showed significantly lower scores in comparison to the normative data for the Italian population16 apart from emotional wellbeing. Participants’ role limitation due to physical problems was particularly impaired, followed by role limitation due to emotional difficulties. The SF-36 role limitation domain evaluates the perception of restriction in people’s own job and daily activities as result of their physical health (RP) or emotional problems (RE), such as anxiety or depression. The restriction is assessed by the SF-36 in term of perceived effort, amount of time spent, quality and efficiency in carrying out work or previous activities.\n\nWe found no correlation between the role limitation scores and the baseline characteristics considered in this study. A prolonged time to recovery following COVID-19 infection as well as the difficulty to access pulmonary and vocational rehabilitation might have contributed to this delay in returning to previous roles, independently from the baseline variables being considered.\n\nCognitive difficulties, particularly memory and concentration deficits, have been described in the spectrum of the post COVID-19 persistent symptoms.5 Although our study did not include any cognitive evaluation, the presence of cognitive difficulties and the presence of mental fatigue could have certainly affected the efficiency in carrying out tasks.25 The restrictions imposed by the policies to contain the COVID-19 pandemic might have been an additional barrier for functioning in the social context. It is relevant in this regard that two thirds of the patients were below 65 years-old and hence in the working age range so that their delayed return to work could have caused a significant financial loss for the patients themselves, their families and employers.\n\nA lower physical functioning scale significantly correlated to a higher CIRS as well as a poorer performance in the sit to stand test, suggesting that people with a higher burden of co-morbilities and who experienced a more pronounced physical weakness during their hospital admission, found it more difficult to resume their performance in daily activities.\n\nSurprisingly, we found age positively correlated to physical functioning and vitality. We argue that younger patients might have experienced a bigger change in comparison to their premorbid level of functioning, hence with a more pronounced effect on their perceived quality of life.\n\nA lower level of functioning and impaired performance in activities of daily life in COVID-19 survivors has been recently described as well as the benefit of an early inpatient rehabilitation intervention.5\n\nSimilarly to our study, Chen et al17 describe HRQL at 1-month post COVID-19 reporting SF-36 sub-scores (RP, SF and RE) significantly lower than in the Chinese population norm. Interestingly the scores reported by these authors are higher in comparison to the scores of our population across all the sub-scores, however the domains more affected are analogous to our findings. We wonder if the younger age of their population in comparison to ours (mean age 47.2 versus 60 years old) might reflect a less severe presentation of the COVID-19 related illness, hence a more limited impact on HRQL.\n\nOther authors6 have reported the results of the SF-36 questionnaire administered to elderly (over 65 year) COVID-19 survivors who underwent 6-week pulmonary rehabilitation, showing an improvement of the HRQL along with pulmonary function. In this study, it is unclear the time when the baseline SF-36 was administered making the findings difficult to compare.\n\nAbout a third of the patients in our study scored in the range for anxiety and depression, similarly to the patients described by Mazza et al.26 Anxiety and depression scores did not correlate to any of the SF-36 domains. Although this lack of correlation could be likely due to the limited sample size, it is also possible that anxiety and depression might have improved following people returning home and that other kinds of emotional difficulties such as feelings of frustration or isolation, rather than anxiety or depression, could have impacted on their return to activities and roles and on the perception of emotional wellbeing.\n\nOur study’s main limitation is the small sample size, which does not allow strong conclusions or generalizations. Furthermore, the Nave Ospedale unit admits a specific group of patients discharged from the acute setting, including those with mild-moderate symptoms requiring further social isolation in absence of alternative accommodation. This suggests a possible social background bias as patients who are more socially disadvantaged may have greater difficulty in finding an alternative suitable environment and therefore may be more likely to be admitted.\n\nMore in-depth questions regarding patients’ jobs and activities on top of the SF-36 could have given a broader insight as well as repeated evaluations of the SF-36 to monitor changes of the HRQL over time.\n\n\nConclusions\n\nWe describe the severe impact across the physical and psychological domains of HRQL in COVID-19 survivors about one month following their discharge from acute hospitals receiving no support from rehabilitation community services. Muscle weakness and the number of co-morbidities at the time of discharge appeared to be associated with lower physical functioning. Interestingly younger patients were more affected in their perceived physical functioning, as well as in vitality. Despite the small sample size, our study clearly highlights the substantial difficulties that COVID-19 survivors experience after their hospital discharge and stimulates a reflection on the individual and social cost of their delayed return to previous roles and jobs. We suggest that dedicated community-based pulmonary and vocational rehabilitation might not only improve these patients’ outcome but could also be socially and financially strategic.\n\n\nData availability\n\nZenodo: Health Related Quality of life in CoViD 19 survivors discharged from acute hospitals Results of a Short-Form 36-ltem survey. https://doi.org/10.5281/zenodo.4540215.27\n\nThis project contains the following underlying data:\n\n- Raw survey data in .xlsx file\n\nData are available under the terms of the Creative Commons Attribution 1.0 Generic (CC BY 1.0).", "appendix": "Acknowledgements\n\nThe authors acknowledge Ms Martina Leggio for her valuable administrative support.\n\n\nReferences\n\nCOVID-19 situation update worldwide, as of week 9, updated 11 March 2021. European Centre for Disease Prevention and Control. Accessed March 13, 2021. Reference Source\n\nNabavi N: Long covid: How to define it and how to manage it. BMJ. Published online September 7, 2020; m3489. PubMed Abstract \| Publisher Full Text\n\nAnxiety and depression in COVID-19 survivors: role of inflammatory and clinical predictors \| Elsevier Enhanced Reader. DOI: 10.1016/j.bbi.2020.07.037\n\nHalpin SJ, McIvor C, Whyatt G, et al.: Postdischarge symptoms and rehabilitation needs in survivors of COVID-19 infection: A cross-sectional evaluation. J Med Virol. n/a(n/a). PubMed Abstract \| Publisher Full Text\n\nCarf’ A, Bernabei R, Landi F, et al.: Persistent Symptoms in Patients After Acute COVID-19. JAMA. 2020; 324(6): 603–605. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBelli S, Balbi B, Prince I, et al.: Low physical functioning and impaired performance of activities of daily life in COVID-19 patients who survived the hospitalisation. Eur Respir J. Published online January 1, 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu K, Zhang W, Yang Y, et al.: Respiratory rehabilitation in elderly patients with COVID-19: A randomized controlled study. Complement Ther Clin Pract. 2020; 39: 101166. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDijkstra-Kersten SMA, Kok L, Kerckhoffs MC, et al.: Neuropsychiatric outcome in subgroups of Intensive Care Unit survivors: Implications for after-care. J Crit Care. 2020; 55: 171–176. PubMed Abstract \| Publisher Full Text\n\nGandotra S, Lovato J, Case D, et al.: Physical Function Trajectories in Survivors of Acute Respiratory Failure. Ann Am Thorac Soc. 2019; 16(4): 471–477. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHopkins RO, Weaver LK, Collingridge D, et al.: Two-year cognitive, emotional, and quality-of-life outcomes in acute respiratory distress syndrome. Am J Respir Crit Care Med. 2005; 171(4): 340–347. PubMed Abstract \| Publisher Full Text\n\nSpruit MA, Singh SJ, Garvey C, et al.: An Official American Thoracic Society/European Respiratory Society Statement: Key Concepts and Advances in Pulmonary Rehabilitation.2013; 188: 17. PubMed Abstract \| Publisher Full Text\n\nPuhan MA, Gimeno-Santos E, Cates CJ, et al.: Pulmonary rehabilitation following exacerbations of chronic obstructive pulmonary disease. Cochrane Database Syst Rev. 2016; 2016(12). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYang F, Liu N, Hu JY, et al.: Pulmonary rehabilitation guidelines in the principle of 4S for patients infected with 2019 novel coronavirus (2019-nCoV). Zhonghua Jie He He Hu Xi Za Zhi Zhonghua Jiehe He Huxi Zazhi Chin J Tuberc Respir Dis. 2020; 43(3): 180–182. PubMed Abstract \| Publisher Full Text\n\nPost-COVID-19 global health strategies: the need for an interdisciplinary approach. Aging Clin Exp Res. Published online June 11, 2020: 1–8. Publisher Full Text\n\nWare JE, Davies AR, Donald CA: Conceptualization and Measurement of Health for Adults in the Health Insurance Study: Vol. V, General Health Perceptions.Published 1978. Accessed August 5, 2020. Reference Source\n\nApolone G, Mosconi P: The Italian SF-36 Health Survey: Translation, Validation and Norming. J Clin Epidemiol. 1998; 51(11): 1025–1036. Publisher Full Text\n\nMahler DA, Mackowiak JI: Evaluation of the Short-Form 36-Item Questionnaire to Measure Health-Related Quality of Life in Patients With COPD. CHEST. 1995; 107(6): 1585–1589. PubMed Abstract \| Publisher Full Text\n\nChen K-Y, Li T, Gong F-H, et al.: Predictors of Health-Related Quality of Life and Influencing Factors for COVID-19 Patients, a Follow-Up at One Month. Front Psychiatry. 2020; 11: 668. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCurci C, Negrini F, Ferrillo M, et al.: Functional outcome after inpatient rehabilitation in post-intensive care unit COVID-19 patients: findings and clinical implications from a real-practice retrospective study. Eur J Phys Rehabil Med. Published online January 4, 2021. PubMed Abstract \| Publisher Full Text\n\nLinn BS, Linn MW, Gurel L: Cumulative Illness Rating Scale. J Am Geriatr Soc. 1968; 16(5): 622–626. PubMed Abstract \| Publisher Full Text\n\nMunari AB, Gulart AA, Dos Santos K, et al.: Modified Medical Research Council Dyspnea Scale in GOLD Classification Better Reflects Physical Activities of Daily Living. Respir Care. 2018; 63(1): 77–85. PubMed Abstract \| Publisher Full Text\n\nA multicentre validation of the 1-min sit-to-stand test in patients with COPD \| European Respiratory Society. Accessed May 19, 2020. Reference Source\n\nZigmond AS, Snaith RP: The Hospital Anxiety and Depression Scale. Acta Psychiatr Scand. 1983; 67(6): 361–370. Publisher Full Text\n\nWare JE, Sherbourne CD: The MOS 36-item short-form health survey (SF-36). I. Conceptual framework and item selection. Med Care. 1992; 30(6): 473–483. PubMed Abstract\n\nMacdonald E, Middleton J, Lalloo D, et al.: Safely returning clinically vulnerable people to work. BMJ. 2020; 370: m3600. PubMed Abstract \| Publisher Full Text\n\nMazza MG, De Lorenzo R, Conte C, et al.: Anxiety and depression in COVID-19 survivors: Role of inflammatory and clinical predictors. Brain Behav Immun. Published online July 30, 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSaverino A: Health Related Quality of life in CoViD 19 survivors discharged from acute hospitals Results of a Short-Form 36-ltem survey [Data set]. Zenodo. 2021. Publisher Full Text" }	[ { "id": "151399", "date": "28 Sep 2022", "name": "Selmin Kose", "expertise": [ "Reviewer Expertise Child health and diseases nursing" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript is a current and important subject. Only, I have some minor recommendations as following:\n\nThis sentence \"COVID-19 survivors particularly the ones of working age may need support for resuming their premorbid level of functioning and returning to work\". should be written more clearly.\n\nAnxiety and depression words should be added for the key words.\n\nITU=What does it mean?\n\nAbbreviations should be explained such as COPD\n\nIn the method, the scales can be given with separate titles.\n\nTable 2 should include p and t values\n\nThe year of the 22th reference should be checked\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "168004", "date": "20 Jun 2024", "name": "Carlos Laranjeira", "expertise": [ "Reviewer Expertise Mental Health", "psychometrics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe material is interesting and the topic is timely and relevant. The method seems to have been followed faithfully and the authors were well-positioned to conduct the analysis. Despite these positives, in my view, the paper needs more work before it could be indexed and I have made some specific suggestions below:\nThe literature addressed is not described accurately so far as I can see. Relevant literature should be presented more deeply in order to support the research problem. Please consider: Figueiredo et al., (20221) & Mitrović-Ajtić et al., (20222).\n\nFurther, there is no clear distinction between manuscript sections in terms of the content they report. First, I suggest dividing the section \"INTRODUCTION\" into three components, respectively introduction (explain the general argument of the paper, without going into specific details) background (situate the study concepts within the context of extant knowledge, discuss the international relevance of the concepts) and purpose, creating greater clarity in the analysis of the reader. What is the study's biggest contribution? The contribution should be clearly stated in the introduction.\n\nAs a reader, I was a bit confused with regard to the purpose addressed in this study. Please provide the specific questions/hypotheses under study, it would be useful for the readers.\nMethods:\nIn your methods section, say that you used the STROBE cross-sectional reporting guidelines.\n\nProvide a clear description of the method used to select the participants for the study. A rationale is needed for the sampling procedure used.\n\nSome subjects refused to participate. Response rate?\n\nThe ethical aspects in collecting data are not specifically clarified, independently of the voluntary nature of the subject´s participation and the approval by the local IRB, variables such as the offer of incentives to participate (how participants were recruited and whether they were compensated for participation), sharing and use of data are not patent.\n\nPlease provide some psychometric properties of used instruments (Italian versions). For better readability list the instruments using bullets.\n\nThe description of the sample (table 1) should be moved to the results section.\n\nResults:\nTable 2 should include p and t values.\n\nHow did the authors handle missing data?\n\nDiscussion:\nSome of the contributions that are highlighted here could be flagged in the introduction for a more consistent narrative throughout the paper. Discussion can be improved if questions/hypotheses will be outlined in the introduction, then answered suitably in this section, and the results interpreted appropriately. I believe there should be better integration of the results with the existing literature.\n\nA stronger discussion of implications for future research and potential intervention work is needed.\n\nChecklist for style:\nThe manuscript will serve a broad audience of students, researchers, and practitioners, however, the manuscript needs to be carefully and attentively proofread. The quality of writing should be reviewed for language and grammar..\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-282
https://f1000research.com/articles/9-1095/v1	04 Sep 20	{ "type": "Software Tool Article", "title": "PUblications Metadata Augmentation (PUMA) pipeline", "authors": [ "Oliver W. Butters", "Rebecca C. Wilson", "Hugh Garner", "Thomas W. Y. Burton", "Rebecca C. Wilson", "Hugh Garner", "Thomas W. Y. Burton" ], "abstract": "Cohort studies collect, generate and distribute data over long periods of time – often over the lifecourse of their participants. It is common for these studies to host a list of publications (which can number many thousands) on their website to demonstrate the impact of the study and facilitate the search of existing research to which the study data has contributed. The ability to search and explore these publication lists varies greatly between studies. We believe a lack of rich search and exploration functionality is a barrier to entry for new or prospective users of a study’s data, since it may be difﬁcult to ﬁnd and evaluate previous work in a given area. These lists of publications are also typically manually curated, resulting in a lack of rich metadata to analyse, making bibliometric analysis difﬁcult. We present here a software pipeline that aggregates metadata from a variety of third-party providers to power a web based search and exploration tool for lists of publications. Alongside core publication metadata (i.e. author lists, keywords etc.), we include geocoding of ﬁrst authors and citations in our pipeline. This allows a characterisation of a study as a whole based on common locations of authors, frequency of keywords, citation proﬁle etc. This enriched publications metadata can be useful for generating project impact metrics and web-based graphics useful for public dissemination. In addition, the pipeline produces a research data set for bibliometric analysis or social studies of science.", "keywords": [ "Longitudinal birth cohort", "Bibliography", "Bibliometrics", "ALSPAC" ], "content": "Introduction\n\nCohort studies collect, generate and distribute huge amounts of longitudinal data for health, social and economic research based on a defined group of people over an extended period of time (often many years). Birth cohort studies begin at birth (or sometimes before) and often continue over the course of their participants’ entire lifetime. The UK is home to many cohort studies and several birth cohort studies, including some that have been running for decades (e.g. the National Survey of Health and Development (NSHD), which started in 19461).\n\nTypically researchers can apply for, and access, these data sets once various relevant governance conditions have been met2.\n\nStudies often keep track of the publications that have arisen from the data they have given to researchers for project monitoring purposes and to report back to their funder(s). The length of these lists of publications is sometimes used as a crude metric of the research outputs or impact for the study.\n\nThe CLOSER (Cohort & Longitudinal Studies Enhancement Resources) consortium (https://www.closer.ac.uk) comprises eight UK birth cohort studies and is used here as an illustration of typical birth cohort studies. Each of the studies holds a list of their publications on their respective public facing websites. The specific purpose, functionality and user interface of these lists varies from study to study. Some studies have publications lists that are comprised exclusively of peer-reviewed journal articles, others have a much broader remit and include a variety of other written outputs, e.g. books, reports, conference proceedings, media examples etc. The way this data is presented varies greatly, ranging from downloadable static PDF files, through static lists on web pages split by year, to interactive web pages.\n\nThe search functionality of each study also varies greatly, with the static page approach offering no other way to search than a browser-based free text search on the rendered text available on a given page. Where only a subset of publications is shown (e.g. if it is split by year), or where rich metadata is missing (e.g. if no keyword or abstract text is available/rendered), it is difficult or impossible to search for given terms. Some studies have a web form which allows a free text search on a database across author, journal, title and abstract text, split by year. One study has an advanced searching capability letting users search on author, subject, article type, as well as free text searching on title, abstract etc. None of the CLOSER studies have any kind of metrics kept alongside their publication lists, e.g. citations.\n\nWe consider the lack of a comprehensive publication search and exploration facility a barrier to entry for researchers unfamiliar with a study. When presented with potentially thousands of publications it can be difficult to find existing publications in a given research area, and when relevant publications have been found, lack of usage information (e.g. in the form of download statistics, citation counts etc) can make it difficult to prioritise reading lists. It is possible that some potential users of a study’s data fall at this first hurdle and do not proceed with an application for data to the study. This could have an effect on the overall impact of the study (since less new research is done) and in the case of studies that charge for data, it will have a direct financial implication. A similar scenario may occur if a researcher doesn’t find a relevant publication and applies for data to carry out a project that has already been conducted.\n\nIn addition to the difficulty of searching and exploring data, the lack of good metadata makes it impossible to do bibliometric analysis on a study’s publications. This means questions such as ’where are all the first authors based?’ or ’are there trends in subject areas over time?’ can only be addressed either anecdotally, or with significant manual input (see e.g. 3).\n\nThis article describes an open source software pipeline (PUMA -PUblications Metadata Augmentation pipeline) which takes a list of publications and augments it with metadata from a selection of third-party metadata providers. This augmented metadata set has two distinct use cases: 1) bibliometric analysis and 2) providing a web based searching and exploration tool. Examples of the potential bibliometric analyses possible with this augmented metadata include: calculating the total number of citations a study has generated, characterising a study based on the keywords of its publications, highlighting the geographic or institutional distribution of first authors, the variety of authors, assessing which journals are published in most frequently, how each of these metrics is changing over time, as well as a variety of other uses. We demonstrate some of these bibliometric uses and a web based exploration tool based on the augmented metadata set provided by PUMA in this article.\n\nWhile the exemplar publications list used here is a U.K. birth cohort, this pipeline could be applied to almost any research study or project that has a list of publications with a rich set of persistent identifiers, particularly in the biomedical domain.\n\nThere are several well established bibliography management tools in which users can manually curate their own bibliographies and easily use them to add formatted references to their written work (see https://en.wikipedia.org/wiki/Comparison_of_reference_management_software for a reasonable list). These include proprietary tools such as EndNote and Mendeley, as well as open source tools like Zotero. A common feature among them is to automatically incorporate available publication metadata from an external source (such as Web of Science, Scopus, CrossRef and others) into each bibliography item. The wide variety and differing levels of completeness of available metadata means that typically a core set of fields are used (e.g. author list is common, but funding information is not). Also, static fields tend to be used, so an author list is common but a citation count is not. These subsets of all available metadata can typically be exported from the various tools in a variety of formats (e.g. BibTeX, RIS etc). There is little focus on gaining insight from the bibliographies in these software packages beyond grouping by keywords/themes.\n\nThe big three bibliometric metadata hubs (Web of Science, Google Scholar and Scopus) all have web based accounts which allow the curation of lists of journal articles and keeps track of the number of citations each article has. They also offer some basic citation analytics such as h-indexes and i10-indexes.\n\nThe focus of these bibliographic tools (both the online hubs and the software) is for an individual’s own published works, or an individual’s collection of publications which they may want to reference later on. Inbuilt to most of the tools is an automatic publication suggestion mechanism which uses the metadata of existing publications to suggest other publications based on common attributes (e.g. similar author lists or keywords).\n\nThere are several other projects which focus on specific visualisation or analytics of existing metadata sets. SurVis4 creates an interactive web based exploration tool based on a static set of BibTeX metadata files. This allows filtering by author or keywords that exist in the static metadata files.\n\nNetwork analysis of authors, subjects, journals, keywords and citations is another area of development, with tools such as CiteWiz5, PivotSlice6 and VOS Viewer7 featuring analysis and visualistion of clusters, trends over time and in depth querying mechanisms.\n\nThese bibliography management, visualisation and analysis tools variously allow the curation of bibliographies, assist in finding similar articles, and give some insight to static metadata. No single existing tool gives easy access to aggregated and processed non-static metadata from a variety of sources to enable both in depth bibliographic study as well as providing an easy to use (potentially public facing) mechanism to explore publication metadata of a long running study.\n\nModern academic journal articles are typically assigned persistent identifiers when they are published. The aim of these is to give a consistent and long lasting mechanism to refer to them. Often a journal will assign a unique journal-specific identifier to an article which resolves to the article on the journal’s website. In addition to this, a Digital Object Identifier (DOI) is usually assigned. DOIs are the de facto persistent identifier used across the academic journal publishing sector.\n\nDOI resolving services exist to refer users (human and machine) to the relevant journal web page. These resolving services also host a wealth of metadata themselves. The service used to resolve DOIs in this work is the canonical resolver: doi.org (see e.g. the DOI data model -https://www.doi.org/doi_handbook/4_Data_Model.html).\n\nIn addition to doi.org there exist other resolving and metadata services that are domain-specific. These may have more in depth and domain specific metadata beyond that offered by the general data model provided by doi.org. In this work we also make use of the persistent identifiers that the National Center for Biotechnology Information (NCBI) PubMed generates (PubMed IDs-PMID), and the metadata their resolving service provides8. This offers us extra metadata over and above that available from doi.org, although on a subset of all available publications.\n\nThe exemplar list of publications considered in this project is from the Avon Longitudinal Study of Parents and Children (ALSPAC - https://www.bristol.ac.uk/alspac). The ALSPAC began in 1990 and as a consequence of this their publications are relatively modern and there is a good coverage of DOIs. The nature of the research done with ALSPAC data is largely biomedical, which gives a high proportion of publications with PMIDs. ALSPAC reports to have over 2000 publications as of April 2019 (http://www.bristol.ac.uk/alspac/news/2019/bristol-families-co90s.html). For this work we use the cleaned BibTeX list of ALSPAC publications9 described in 10. For a general overview of ALSPAC see 11.\n\n\nMethods\n\nPUMA is built as a pipeline of several discrete stages, the first stage retrieves a list of publications, then subsequent stages add and derive information for each publication in the list before passing it on to the next stage. The end goal of this augmentation stage is a metadata object containing as many metadata items in Table 1 as possible. This is achieved by first retrieving the list of publications from Zotero; adding metadata to it from doi.org, PubMed, and Scopus; geocoding the first author’s institute; and getting citation counts. Once this metadata object has been built PUMA can then do some basic statistics and generate HTML pages to allow exploration and searching. This is explained in detail below, and shown in overview in Figure 1.\n\nThis is a tabular representation of the python dictionary used to store the metadata, the secondary column items are nested under the primary items where present. Metadata source key: Zr=Zoteroraw, S=Scopus, D=doi.org, P=PubMed, Ze=’Extra’ field from Zotero, W=Wikidata, De=Derived. The metadata sources are used in the order they are displayed in the table (left to right), once a value has been found the subsequent sources are not queried.\n\nThe left column shows the sources of data accessed via their APIs, the central column the stages the pipeline with the right column showing input and output of the pipeline.\n\nZotero. Zotero is an online, free-to-use and open-source bibliography manager. It allows publications metadata to be grouped together into user defined libraries. Here we use it to hold the canonical list of unique publications for a given study (ALSPAC in this exemplar). Zotero allows publications metadata to be entered manually (by filling in the fields by hand), semi-manually (by adding e.g. a DOI and it querying external sources), or programmatically using its API.\n\nOnce a publication’s metadata is stored in Zotero it can be updated as required. One particularly useful feature of Zotero is its built in deduplication function. Whilst the exemplar list of publications used here is clean (i.e. there are no duplicates in it), if a new list is used it is likely that this will not be the case. Data cleaning can be done in Zotero, and this is made easier by Zotero pulling in metadata from external sources (Crossref and PubMed for the items here). This can highlight, and enables easy fixing of, errors such as where a DOI has been mistyped and points to an incorrect publication.\n\nThe PUMA pipeline begins by using the Zotero API (v3) to get the library for the study. This is presented as a series of JSON files (one per publication in Zotero), containing all the metadata held on a given publication. These are downloaded and cached locally by the pipeline.\n\nWhile all of the metadata is downloaded from Zotero, the PUMA pipeline disregards most of it as it does not map to the final metadata object well. The most important fields used from the Zotero metadata are the DOI and PMID, since these are the identifiers used to query external metadata providers. DOI has a native field in Zotero, but PMID does not and is stored as a key-value pair in the Zotero ’extra’ field. There are a small number of fields where if the metadata is missing from doi.org and PubMed, then the metadata is used from Zotero. Where there is a direct match to the native Zotero data type (e.g. title) that is used, where there is not a match (e.g. Zotero doesn’t have an affiliation field for authors) then a key-value pair is used in the ’extra’ field. This is outlined in Table 1.\n\ndoi.org. The pipeline then cycles through the list of publications, and where a DOI is present in the metadata from Zotero, it queries the doi.org API with it. If it is a valid DOI then doi.org will return a JSON file containing all the metadata it holds on this publication, which is cached locally.\n\nPubMed central. If the Zotero metadata contains a PMID then the pipeline will then query the PubMed central API to get any extra metadata. The resulting XML file is cached for later use. The metadata available from PubMed is richer than that available via doi.org, including biomedical domain specific fields such as MeSH headings.\n\nScopus. Scopus is then queried via it’s API. The query is first tried with a PMID, then if no value is found the query is repeated using the DOI as the identifier.\n\nThe use of Scopus data has some constraints on it depending on the context in which it is used. The most relevant condition here is that where citation counts are displayed on a website they must link back to the relevant publication in Scopus, and must be updated at least weekly (https://dev.elsevier.com/tecdoc_attribution_ scopus.html).\n\nMerging DOI, PMID & Scopus metadata. As noted earlier, the metadata from doi.org and PubMed will contain different fields. Moreover, the same field may have different names in the two sources. In order to merge the metadata in a meaningful way we developed a mapping from each of the relevant fields to what we consider the local canonical version. In some cases our mappings required several fall-backs, e.g. the date of a publication in PubMed has six different places that it could be specified. This is due to a combination of the PubMed schema changing over time, the completeness of the data when it is input into PubMed, and genuine different relevant dates e.g. date published online and data published in print.\n\nThe mapping is done into our core set of fields (see Table 1) for each metadata source. Our mapping process initially creates a simple metadata object based on the Zotero ID, DOI and PubMed ID. Into this metadata object it then copies the relevant fields from the DOI, PMID and Scopus metadata.\n\nGeocode. We assume the first author of the publication is the primary author, then we attempt to assign a canonical institute to them. This assignment is done by using a manually built lookup table which initially tries to use the email address of the first author, then if that fails, the postal address. In order to get consistent geographical information of a publication we take a university to be the smallest unit (i.e. two different departments at the same university will not be distinguished in the geocoding). The reason behind this is that there is very little consistency between publications on how department addresses are formatted. The same strategy is used for hospital departments and companies. Our definition of what a canonical institute name is is based on how it appears in wikidata (https://www.wikidata.org).\n\nFor the email address based matching we attempt to match exactly the domain part of the email address to a canonical institute (e.g. someone@ncl.ac.uk gets mapped to Newcastle University). Email addresses that are generic or personal (e.g. someone@gmail.com) are ignored.\n\nIf there is no matching email address then we attempt to match the postal address. The lookup table has multiple entries for several organisations where authors use non-canonical names e.g. ’Newcastle University’ and ’The University of Newcastle’ both map to Newcastle University.\n\nSince a publication list may go back many years, there may be institutes that no longer exist (perhaps having been renamed, merged with other institutes or shut down entirely). The lookup table therefore has several entries of now defunct institutes which are mapped to from email addresses and postal addresses.\n\nOnce we have the canonical name for an institute we use the wikidata SPARQL API to get the institute’s geolocation, town and country (wikidata properties: P625, P131 and P17, respectively). In some cases the first author’s institute may be a large multinational or distributed organisation, in which case we use the head quarters location as defined on wikidata (property P159). If any of this data does not exist on wikidata we try to add it.\n\nData quality. The nature of the manual curation of a list of publications can lead to some missing information and errors. This ranges from there not being any persistent identifiers present, to multiple copies of the same publication being present in different forms, e.g. a preprint and the final version. In order to address these data quality issues we built an interface to assist further cleaning of the metadata, there are two main facets to this interface: highlighting issues and making fixing issues easier.\n\nThe interface is a large HTML table with a row for each publication and columns for the status of relevant attributes. Where a value of an attribute is useful in the data cleaning process it is displayed (e.g. DOI and PMID), where the presence of an attribute is more useful than its value (e.g. first author) then just an indication of it’s presence is given. Missing attributes are colour coded to make them easy to see, and the table can be sorted by value/presence of attributes. Where, for example, a PMID is missing, the relevant table cell is coloured orange and there is a clickable link which queries PubMed for this publication based on its DOI or title. Similar approaches are available to find DOIs via PubMed and Scopus. Where this provides missing IDs (DOI or PMID) they can be added to Zotero and the pipeline rerun.\n\nSome metadata may not be present in the external providers metadata for some publications -even with the correct DOIs and PMIDs in Zotero. In this case the metadata can be used directly from Zotero for a small number of fields as indicated in Table 1. As with the DOI and PMID case above, the missing metadata will be highlighted orange in the table, and once it has been added to Zotero the pipeline will need to be run again.\n\nThere are some derived metadata items that, if missing, will be highlighted in red; this indicates that a setting in the pipeline or a local configuration file is causing the problem. An example would be if a first author institute is found in the source metadata, but there is no matching entry in the institute look up file then a canonical institute cannot be set. The lookup file needs to be updated and the pipeline rerun in this case.\n\nSimple analyses. The pipeline then does some simple processing of the metadata so it can be used for reporting and which feeds into the generated web pages (see below). It outputs (as a CSV file) the frequency of the authors (separately the full author list and first author only), the first author’s institute and the journal the publication appeared in.\n\nThe keywords, title and abstract text in a publication all serve to give an overview of the content. The keywords are sometimes from a controlled vocabulary, e.g. Medical Subject Headings (MeSH). The titles and abstracts having more free text offer the ability to be more descriptive. From a searching perspective the greater freedom with abstracts makes them more searchable/findable12. To derive some meaning from all of the available text in all of the publications from a study, the pipeline calculates the frequency of each word in the keywords, titles, and abstracts. To process the text it converts all text to lower case and removes all punctuation. It then takes out the name of the study, so in this case the exact phrase \"Avon Longitudinal Study of Parents and Children\", and variations of, but individual components are kept if they were used outside of that context e.g. if ’parents’ is used in a different sentence. Then common words such as the, and etc are disregarded. Then the Python Natural Language Tool Kit13 is used to lemmatize each word into its base component. With this clean set of words the pipeline then calculates the frequency of each. It also does this broken down by year, so it is possible to see how trends in research areas change over time in a long running study.\n\nGenerated web pages. The pipeline generates static HTML pages which allow the search and exploration of the augmented metadata sets. These pages include filtering by year and by keywords, and visualisations of some of the metadata. The static HTML pages are completely encapsulated, meaning that they can be viewed without the need for a web server etc. As such, PUMA can run locally to generate the data and statistics, and then the HTML files used to explore it.\n\nFigure 2, Figure 3 and Figure 4 show example plots from the generated web pages, with live versions available at https://ollybutters.github.io/puma/alspac/.\n\nThe x-axis is truncated at 200 citations as there are a small number of publications disparately spread above this.\n\nThe pipeline is written in Python 3 and is available from GitHub (https://github.com/OllyButters/puma). Some prerequisite Python libraries are required to run the PUMA pipeline, these are described on the wiki at https://github.com/OllyButters/puma/wiki and in the requirements.txt file in the source folder.\n\nThe behaviour of the pipeline (which API keys to use, date ranges, colour schemes, caching behaviour etc) is controlled by a configuration file. A sample configuration file is available in the config directory, with guidance on how to populate it at https://github.com/OllyButters/puma/wiki/Configuration. The pipeline has been developed in linux environment, and can be run from the command line by calling the papers.py file in the source directory. This assumes a config.ini file in the config directory, if a different file name is used then it can be specified when running the pipeline with the config option: papers.py --config-file-name.ini. We have also run PUMA in a Windows 10 environment with a minimum of python 3.6.\n\nThe pipeline is designed so that it can be rerun regularly, running as a regular CRON job for example. Metadata is cached locally wherever possible, making subsequent pipeline runs much quicker after the initial run. This is possible as the metadata from doi.org and PubMed is very stable, so changes are rare to an individual publication’s metadata. The citation counts are cached for as long as is specified in the configuration file, being updated as required.\n\n\nUse case\n\nWhile the intention of this article is to describe the pipeline and the work flow developed to ingest metadata into it we also show some example outputs of the pipeline without interpretation.\n\nWe imported the ALSPAC BibTeX data into a new collection in a new group library in Zotero, giving the coverage of fields as outlined in 10. The PUMA pipeline works best with at least one of DOI and PMID for each publication, coverage of these fields in the source metadata is outlined in Table 2. See Underlying data for a list of the references used14.\n\nFor this initial metadata set the pipeline achieved the augmented metadata coverage outlined in Table 3. Where there are gaps in this metadata it is mostly due to actual missing metadata in the source systems, however the incompleteness in the geocoded metadata is due to a combination of authors using a consortium name as their affiliation, or the metadata containing only a fragment of their address. These could easily be manually addressed with the ’extra’ field in Zotero, however since the purpose of this article is to outline the PUMA pipeline and not to strive for a 100% coverage of the metadata, we have not added any ’extra’ metadata to Zotero.\n\nFigure 2 shows the number of publications published per year for ALSPAC from 1989 to 2015. This is the most basic information from the pipeline, and is already information that is easily available to the studies.\n\nTable 4 shows some basic study level citation calculations. As noted above, the incompleteness of the metadata will impact the numbers here, specifically ALSPAC has 98% Scopus coverage, meaning all the citation numbers in Table 4 will likely be slightly under-reported.\n\nFigure 3 shows the profile of citation counts for ALSPAC publications with a citation count less than 200.\n\nUsing the geolocation data generated from the pipeline we can plot a choropleth map of countries first authors are based in. Figure 4 shows the first authors location for 98% of the publications. Again, this is affected by the coverage of the metadata.\n\nTable 5 shows the result of a frequency analysis of lemmatized keywords, title text and abstract text of all of the publications that have relevant metadata. This metadata can be further broken down by year to show study changes over time.\n\nThe numbers in parentheses are the count.\n\n\nDiscussion\n\nOne limitation to this work, which is difficult to address, is the completeness of the source list of publications. It is common for cohort studies to ask researchers to inform them when they publish their research based on the study’s data. This request is not always complied with, so the source lists of publications are prone to being incomplete. This will have an impact on the insights the PUMA pipeline can generate, with some aspects just under-reporting (e.g. the total citation count) while others may give a misleading picture if there is a systematic reason for the missing publications (e.g. the frequency of keywords in a study will be misleading if all publications from a field are missing).\n\nOne of the key assumptions we have made is that the first author is the primary author for the publication. This does vary across different scientific disciplines - it may be that the first author is the one who did the bulk of the work, or that they wrote up the majority of the publication, or they just appear first alphabetically. While this will not have an effect on the publication-level statistics (e.g. how many citations it has), it may have an effect on where we have assigned a geographic location.\n\nLinking on author name is also problematic when multiple authors have the same name, or where there are multiple spellings of a given name. This can occur where names have been converted to e.g. ASCII on their way into metadata records. Another instance is where a name is sometimes hyphenated and others not (in this exemplar data set there exists entries for Davey Smith and Davey-Smith). It is important not to place too much emphasis on citations and to not treat them as an exact value. While it is easy to measure how many publications are cited in a single publication, it is difficult to establish the inverse - i.e. how many publications in all the literature cite a given publication. This is due to the completeness of the source literature which is used to calculate the incoming citations, which means that different providers of citation counts will likely give different answers (see 15 and 16).\n\n\nFuture work\n\nThe modular nature of the pipeline means that it is straightforward to add different data sources. One source that we plan to add is Altmetric, which tracks mentions of publications in the media (including social media) and links these back to a DOI. We also plan to link directly with Crossref (using their API) to pull in a richer set of metadata.\n\nSome of the modern PubMed metadata, and a lot of the Crossref metadata, include information on grants (increasingly with a grant reference code). This would allow us to investigate who the major funders of users of the data are.\n\n\nData availability\n\nZenodo: ALSPAC peer reviewed publications 1989-2015. http://doi.org/10.5281/zenodo.227678514.\n\nThis project contains the list of publications from the Use case. All other metadata is pulled in from external APIs at run time.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nSoftware availability\n\nSource code available from: https://github.com/OllyButters/puma.\n\nArchived source code at time of publication: http://doi.org/10.5281/10.5281/zenodo.397110217.\n\nLicense: GNU General Public License v3.0.", "appendix": "References\n\nKuh D, Pierce M, Adams J, et al.: Cohort profile: updating the cohort profile for the MRC National Survey of Health and Development: a new clinic-based data collection for ageing research. Int J Epidemiol. 2011; 40(1): e1–e9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMurtagh MJ, Blell MT, Butters OW, et al.: Better governance, better access: practising responsible data sharing in the METADAC governance infrastructure. Hum Genomics. 2018; 12(1): 24. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBarbosa SDJ, Silveira MS, Gasparini I: What publications metadata tell us about the evolution of a scientific community: the case of the Brazilian human–computer interaction conference series. Scientometrics. 2017; 110(1): 275–300. Publisher Full Text\n\nBeck F, Koch S, Weiskopf D: Visual Analysis and Dissemination of Scientific Literature Collections with SurVis. IEEE Trans Vis Comput Graph. 2016; 22(1): 180–189. PubMed Abstract \| Publisher Full Text\n\nElmqvist N, Tsigas P: CiteWiz: A tool for the visualization of scientific citation networks. Inf Vis. 2007; 6(3): 215–232. Publisher Full Text\n\nZhao J, Collins C, Chevalier F, et al.: Interactive Exploration of Implicit and Explicit Relations in Faceted Datasets. IEEE Trans Vis Comput Graph. 2013; 19(12): 2080–2089. PubMed Abstract \| Publisher Full Text\n\nvan Eck NJ, Waltman L: Software survey: VOSviewer, a computer program for bibliometric mapping. Scientometrics. 2010; 84(2): 523–538. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNCBI Resource Coordinators: Database resources of the national center for biotechnology information. Nucleic Acids Res. 2018; 46(D1): D8–D13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nButters O, Ismail A, Thompson S, et al.: ALSPAC peer reviewed publications 1989–2015 . 2018. Publisher Full Text\n\nButters O, Ismail A, Thompson S, et al.: Generation of a cleaned dataset listing Avon Longitudinal Study of Parents And Children peer-reviewed publications to 2015 [version 1; peer review: 2 approved]. Wellcome Open Res. 2018; 3: 161. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBoyd A, Golding J, Macleod J, et al.: Cohort Profile: The ‘Children of the 90s’ - the index offspring of the Avon Longitudinal Study of Parents and Children. Int J Epidemiol. 2013; 42(1): 111–127. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVan Kasteren Y, Williams PAH, Maeder A: Identifying emerging trends in medical informatics: A synthesis approach. Stud Health Technol Inform. 2017; 235: 506–510. PubMed Abstract \| Publisher Full Text\n\nBird S, Loper E, Klein E: Natural Language Processing with Python. O’Reilly Media Inc., 2009. Reference Source\n\nButters O, Ismail A, Thompson S, et al.: ALSPAC peer reviewed publications 1989–2015. 2018. http://www.doi.org/10.5281/zenodo.2276785\n\nMingers J, Leydesdorff L: A review of theory and practice in scientometrics. Eur J Oper Res. 2015; 246(1): 1–19. Publisher Full Text\n\nMartín-Martín A, Thelwall M, Orduna-Malea E, et al.: Google Scholar, Microsoft Academic, Scopus, Dimensions, Web of Science, and OpenCitations’ COCI: a multidisciplinary comparison of coverage via citations. arXiv. 2004.14329, 2020. Reference Source\n\nButters O, Wilson B, Garner H, et al.: OllyButters/puma: v1.2. 2020. http://www.doi.org/10.5281/zenodo.3971102" }	[ { "id": "72761", "date": "26 Nov 2020", "name": "Leslie McIntosh", "expertise": [ "Reviewer Expertise Informatics", "bibliometrics", "scientometrics", "biomedical informatics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall Comments Improving the search capabilities across studies based on research manuscripts is challenging and important. The authors have built a pipeline to improve the capture of metadata, thus addressing this challenge. The links to the data and software work. Moreover, I replicated the study with the available data, code, and instructions without contacting the authors. While there are many comments below, the majority pertain to improving the article readability. Great work on building this pipeline.\nOpen Science This part of the review focuses on the transparency of the work looking for quality indicators that are important for a good manuscript. While all of these elements are not necessarily required by the publisher, the presence and clarity of them make your work more transparent.\nAuthors One author has an ORCID listed while the others do not. It is best practice to have unique, persistent author identifiers such as an ORCID for each author.\n\nStudy Objective Your Text from the Introduction: This article describes an open source software pipeline (PUMA -PUblications Metadata Augmentation pipeline) which takes a list of publications and augments it with metadata from a selection of third-party metadata providers.\nComment: This technically states what you are doing and is not a clear study objective. A clear, concise, study objective will help in reading and understanding the science (e.g., The purpose of building the software pipeline is to improve search capabilities across cohort studies through augmenting metadata.)\n\nData Availability Statement The DAS is present and links to the Zenodo corpus of papers for the Use Case. You could also state that the other data presented is generated from the software/code.\nCode Availability The code is available as stated in the Software Availability statement and is working as expected.\nFunding Statement This is present with appropriate grant identifiers mentioned. I did not verify the grant IDs.\nEthics Statement When reviewing your manuscript, I could not find the ethical approval of the research. As this appears not to apply to this research, this is acceptable.\nOrganisation and Miscellaneous This section provides critiques based on the readability of the article. Where appropriate, I offer suggestions for making it more consumable to the reader.\nIntroduction\nThe information is great in the introduction, but I found it difficult to read at first. Suggestions for improvement:\nThe first through fifth paragraphs (ending with “The search functionality… paragraph” seems out of place. Perhaps call out the description of the cohort study data just has you have other topics in the introduction (e.g., Existing Tools, Exemplar Publication List) and put it later (see next comment).\n\nThe true introduction seems to start with the sixth paragraph “We consider the lack of a comprehensive publication…” until the ‘Existing Tools’ Section. This section succinctly describes the problem with searching across cohort publications and offers your study objective.\nFrom the sentence beginning with “In this work we also make use of the persistent identifiers that the National Center for…” through the end of the next section appears to have truly started describing your Methods.\n\nMethods\nThe Implementation subsection is a bit challenging to read. It might make it more readable to add labels to Figure 1 so they can be referenced in each section describing it. There are invariably other possible solutions as well.\n\nDescribe Table 1 in more detail before it is referenced.\n\nFigure 3 - the mean and median are hard to see with the yellow and green colours. (You don’t necessarily need to fix this in the paper but maybe in a future iteration of the software.)\nOther\n\nThe keywords for the article should include something related to pipeline development and/or software.\n\nThere are a number of grammatical errors throughout the article.\n\nConsider dropping ‘etc.’ when you are stating examples, especially when using ‘(e.g., )’\nScientific Review\n\nThis section delves into the scientific question(s) presented in the article. I am specifically looking for an interesting scientific rationale for the study as well as supporting evidence to answer the research objective(s).\n\nStudy Objective as stated in article\n\nThis article describes an open source software pipeline (PUMA -PUblications Metadata Augmentation pipeline) which takes a list of publications and augments it with metadata from a selection of third-party metadata providers. This augmented metadata set has two distinct use cases: 1) bibliometric analysis and 2) providing a web based searching and exploration tool. Examples of the potential bibliometric analyses possible with this augmented metadata include: calculating the total number of citations a study has generated, characterising a study based on the keywords of its publications, highlighting the geographic or institutional distribution of first authors, the variety of authors, assessing which journals are published in most fre- quently, how each of these metrics is changing over time, as well as a variety of other uses. We demonstrate some of these bibliometric uses and a web based exploration tool based on the augmented metadata set provided by PUMA in this article.\nThe software ran on my local machine and using the corpus suggested within Zotero, everything replicated. Great job! Working with Zotero does provide a few challenges, but there are notes on the GitHub wiki that walks through those challenges.\n\nThe counts provided Tables 2-5 come from a CSV file generated after running the code. It would be nice to have the files and code for the use case to be shared as well. This not only supports complete reproducibility, it also helps some readers understand the expected outputs without installing the software.\n\nThe Use Case is very nice to have in these types of projects, so I would suggest being clearer with it. What questions can you answer from these data that you could not before? Why is this important?\n\nThe Discussion should loop back to the purpose of the study and restate what you achieve through your work.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "6392", "date": "12 Apr 2021", "name": "Olly Butters", "role": "Author Response", "response": "Thank you for taking the time to read the paper and installing/running it! We think we have addressed all of your comments and have itemised this below. Open Science ORCIDs – All authors have now added ORCIDs. Study objective – Updated the text as suggested. DAS – added an output section with a link to Zenodo with all the output data and some screenshots. Organisation and Miscellaneous Introduction - Substantially restructured the introduction along the lines suggested here and in the other referee's comments. Some content moved into a separate background section and the use case data description moved to the use case section later on. Method Aligned the heading names in the implementation section to the middle column of the figure and highlighted this in the text. Reworded how and where table 1 is referred to. The colours on the plots are mostly configurable in the software, but I agree it is not easy to see that in the diagram. I have added the median value to the citation metrics table to help with this paper. Other Keyword – Added ‘Software pipeline’ Fixed typos and grammatical errors throughout. Got rid of several ‘etc’ Scientific review Glad you got it to run! Table data: Table 2 – The counts come from manually looking at the raw data. Table 3 – The metadata coverage table values are copied from the output HTML. I have added a screen shot in the Zenodo output data of this and it is signposted in the table caption. Table 4 – The citation values are copied from the output HTML. I have added a screen shot in the Zenodo output data and this is signposted in the table caption. Table 5 – Lemmatized word frequencies – Added the full list of lemmatized words to the Zenodo output data and signposted in the caption. Use case – I have updated the text to have a brief discussion about some of the derived data. Discussion – updated the start of the discussion to be more of a summary." } ] }, { "id": "70885", "date": "16 Dec 2020", "name": "Meena Khatwa", "expertise": [ "Reviewer Expertise Primary Qualitative research", "systematic reviews in evidence base policy in public health. Specialist in stakeholder engagement", "qualitative evidence synthesis and thematic analysis. Lecture on MSc module : Introduction to Qualitative Methods." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall comments This was a very interesting article on finding ways to find a user-friendly way to source and manage data. As a systematic reviewer, the processes used are not so dissimilar to how we scope and search for evidence.\n\nOn the whole I found the paper engaging and have only minor suggestions and comments to make. I am a qualitative research who specialised in qualitative evidence synthesis and thematic analysis. Therefore I positioned myself as someone who as a first time researcher would be interested in applying this method.\n\nAbstract Clear and concise but I would have made it more explicit this was project to trial/test PUma.\nIntroduction I understand this project was primarily funded by CLOSER; \"This project was primarily funded by CLOSER, whose mission is to maximise the use, value and impact of longitudinal studies. CLOSER is funded by the Economic and Social Research Council (ESRC) and Medical Research Council (MRC) (grant)\" hence using their data, as you state as an ‘illustration’ (p 3). It would have useful to have compared to another data set to have avoided any conflict of interest or biasness.\n“Some studies have a web form which allows a free text search on a database across author, journal, title and abstract text, split by year. One study has an advanced searching capability letting users search on author, subject, article type, as well as free text searching on title, abstract etc. None of the CLOSER studies have any kind of metrics kept alongside their publication lists, e.g. citations”. (p3)\nThis paragraph needs to make more clarity as it is unclear, what do you mean by one study and what is that study. The information is a bit vague.\n“We consider the lack of a comprehensive publication search and exploration facility a barrier to entry for researchers unfamiliar with a study. When presented with potentially thousands of publications it can be difficult to find existing publications in a given research area, and when relevant publications have been found, lack of usage information (e.g. in the form of download statistics, citation counts etc) can make it difficult to prioritise” pg. three\nThere is a disconnect between this paragraph and the one before – clearer connections between the arguments and rationale here how have the CLOSER studies led to this rationale in this paragraph.\nFor first time users – it would be helpful to define what you mean by, “bibliometric”, as it used quite frequently in the paper.\nExemplar Publication list (p4) You state here the exemplar list of publications considered in this project is from the Avon Longitudinal Study of Parents and Children (ALSPAC). It would really be helpful from the outset if the author could be clear that this was a project undertaken to test the PUMA pipeline there seems to be a switch from study to project – to maintain consistency perhaps refer to one description.\nMethods Page 7 - unclear by what you mean by ‘Use Case’ - what does this mean?\nI found the diagram and definition of each stage very helpful in regards to the process undertaken.\nDiscussion\nIt would be really helpful to know how long it took to use PUma when trialling it for this project. Where there any technical glitches? How user friendly it is, particularly in regards to accessibility (especially for students or first time researchers)?\nFuture work If this is to be utilised by others what will be available as a resource guide for those who want to use it (e.g. training etc.)?\nHeadings and signposting There seem to be three types of headings being used to signpost different sections. At times I found this confusing as the main heading and subheading seem to be same font size and weight (e.g. see discussion the limitations underneath then you have sub subheadings in italics).\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Partly\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "6393", "date": "12 Apr 2021", "name": "Olly Butters", "role": "Author Response", "response": "Thank you for taking the time to review this paper, and we’re very happy to hear you found it interesting! I think we have addressed all of your comments in the text. Below we itemise each one and how we have addressed it. Abstract Added text to be explicit about this being a test of PUMA using a list of publications from a cohort study. Introduction Comparing to another dataset – we found that the amount of work required to generate a clean list of publications was high (see the ALSAPC data paper referenced here). For now, we just wanted to show what the pipeline is. Our plan is to do as you suggest in the future and run the pipeline on a selection of different studies so that we can look in more detail at the differences between them. Cohort study web form searching – tidied up language, and updated text to reflect CLOSER’s increased membership. Restructured the introduction to make this clearer. Added a bibliometrics definition Exemplar publication list Moved this section to the use case section and made it clearer throughout that this data is to show the pipeline working. Tidied use of project/study throughout. Methods Use case - The term use case comes from the F1000 software tool article structure and it's just meant to show an example of the pipeline running. Discussion Outlined in the operation section that it is a rather technical program to run, and also mentioned in limitations section. Added the time it took to run in use case section. Added discussion point about its development. Future work Currently, I'd expect the main users to be at the more technical/development end. The documentation on the wiki covers all aspects of how to run it and was sufficient for Leslie McIntosh to install and replicate the analysis. It would be interesting to partner with some bibliometric analysis researchers to see where the software and training needs to go next, but that will require more funding. Headings Tidied up use of headings to make it easier to follow." } ] } ]	1	https://f1000research.com/articles/9-1095
https://f1000research.com/articles/10-89/v1	10 Feb 21	{ "type": "Research Article", "title": "Exploring the critical points of teaching STEM subjects in the time of COVID 19: the experience of the course \"Microscopy Techniques for Forensic Biology\"", "authors": [ "Elvira Brunelli", "Rachele Macirella", "Rachele Macirella" ], "abstract": "Background: The University was among the first structures to be hit by the health emergency, transferring all its teaching and research activities remotely. It was not easy for teachers and students to find themselves suddenly shifted into different teaching and socializing context. Results: This article describes and analyzes the online teaching experience carried out for the course of Microscopy Techniques for Forensic Biology offered as a part of the Master's degree program in Biology at the University of Calabria (Italy). A cross-sectional survey (pilot study) was designed to investigate the accessibility of distance learning along with an evaluation of adjustments needed for the conversion from offline to online instruction. Particular attention has been paid to learning material and lesson duration, with specific emphasis on practical activities. Conclusions: The author's intent is that of opening a comparison between the strengths and weaknesses that emerged in this experience, highlighting, in particular, how the educational relationship between teacher and student has changed.", "keywords": [ "Covid-19 outbreak", "online learning", "STEM", "Science laboratory", "Classroom climate." ], "content": "Introduction\n\nThe traditional academic and research community asked learners to handle paper-based documents, taking the form of exercise books, notebooks, lecture notes, etc., and a way of transmitting knowledge mainly based on the frontal lessons. The use of modern technologies and e-learning-based culture partially modified the formulas of education (Singh & Thurman, 2019); the use of digital resources is now very diffuse, also representing a useful communication system for the needs of science. According to the European Commission’s directives, relating to the Lifelong Learning Program, which promotes the transformation of education through technology, (https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX%3A52004PC0474), an e-learning service to support didactics (https://24cfu.unical.it/elearning/) has been created at the University of Calabria, Italy. The overall objective envisaged was the enrichment of traditional lessons through the use of network technologies and related paradigms. This e-learning service relies on Massive Open Online Courses (MOOCs) that support distance education, acknowledging the existence of a plurality of academic training offers (Kaplan & Haenlein, 2016). Despite these growing innovations, until now, the most common teaching method in university education remains the face-to-face lessons that immediately enable the teacher and the students to communicate easily and confidently.\n\nFor preventing the expansion of COVID-19, in Italy, restrictive measures were adopted almost immediately, leading to the closure of higher education institutions and the suspension of face-to-face activities on March 5, 2020. Given the medium to the long-term perspective of suspension, teachers were all asked to deliver their lectures via the Internet to ensure education and give students the continuation of their intellectual experience. To guarantee a standard implementation of courses and facilitate both teachers and students, the University of Calabria found an adequate settlement through the choice of the Microsoft Teams platform (©Microsoft Teams Version 1.3.00.3564 for Windows and 2.0.15 for iOS, Microsoft Corporation). In this scenario, the students experienced a sudden and unwanted time and spatial separation, and the lesson delivery shift was not readily accepted. While the focus is mainly placed on the impacts on students, teachers also faced some challenges, including the choice of instructional strategies, learning resources, and assessment methods. Besides, since from a constructivist perspective enhancing interaction with peers is an issue that should be addressed, it is crucial to pay adequate attention to the interactivity during online learning (Huang et al., 2019).\n\nFurthermore, the conversion from an offline to online instruction when courses include the development of specific competencies through practice, as is the case in science, technology, engineering, and math (STEM) courses, required special attention. Although STEM education has drawn increased consideration in the last few years, little attention has been paid on how it should be realized in practice during online learning. Here, we report the online teaching experience for a course in the STEM area, offered as a part of the Master's degree program in biology. The course includes both lessons and practical laboratory sessions delivered for 21 students with a high level of confidence in using digital tools and web-based applications. This paper aims to participate in the dialogue about online education during the pandemic contributing to the definition of the future strategy aimed to overcome the limits of non-traditional learning.\n\n\nMethods\n\nThe platform used for delivering online lessons was Microsoft Teams (©Microsoft Teams Version 1.3.00.3564 for Windows and 2.0.15 for iOS, Microsoft Corporation). The software Teams, like several other applications, allows different modes to hold virtual classes i) pre-recorded audio incorporated in the slide presentation files, ii) recorded classes, allowing students to access on-demand; iii) virtual classes, for students to watch in real-time (Rainbow, 2020). Before beginning their activity, all teachers attended the University's training, coordinated by the office in charge, to become familiar with the platform and acquire basic knowledge on how to use, create, and share resources from the perspective of open education (Bussis & Chittenden, 1970). The same office issued a memorandum addressed to teachers and students, giving the information on software functions and explaining the platform's essential tools.\n\nThe course of \"Microscopy Techniques for Forensic Biology\" is a core course offered as a part of the Master's degree program in biology. It includes 36 hours of lessons and 24 hours of practical laboratory work, reaching a total of six ECTS credits (according to the European Credit Transfer and Accumulation System). The course began on March 16, 2020, and ended on June 08, 2020, and was attended by 21 students. The course was delivered entirely online, holding a virtual class supported by slides projection and integrated with other online support such as pre-recorded video, particularly for laboratory work. A downloadable version of the course program was available from the beginning of the academic year on the website of the degree program in biology. The program comprised the synthetic content of the course, divided into 13 topics. It also included the list of suggested texts (e-books and paper-books) and several online resources that are useful to address each topic. On the contrary, the learning material (i.e., a printable version of slides) was shared on the platform just before the official beginning of the course. The slides provided, organized according to the 13 topics reported in the program, were numbered (Table 1). It was, therefore, possible to compare the material produced at the end of the course.\n\nA cross-sectional survey (pilot study) was designed to investigate the accessibility of distance learning. An online self-administered survey was conducted in June-July 2020. Questionnaires received after July 7, 2020, were excluded from the study. Questionnaires were completed using the online tool Google Forms. Given the aim of the study, the sample size was not a priori determined, and no ethics committee approval was required. None of the participants shared personal information, and the data of the students were anonymous. Participation in the survey did not require approval from an ethics committee as the online questionnaire's completion was on a voluntary basis and anonymous; it was impossible to track the sensitive personal data of all participants (including the IP address). Before starting the questionnaire, a brief description of the study, the declaration of confidentiality and anonymity were furnished to students. Answers were saved by clicking on the button “submit” and the students could suspend the questionnaire at any time.\n\nThe online questionnaire has complied with national and international agreements and regulations, with the Declaration of Helsinki (2000) and the General Data Protection Regulation of the European Parliament (GDPR 679/2016). In the invitation email and the introduction to the survey, we explained research purposes and that the University of Calabria was responsible for data collection and management. We specified that the project and its findings were going to be published in scientific articles. The questionnaire was written in Italian and comprised 16 questions (single-choice questions and one open question); an English translation of the questionnaire is provided in Table 2. For seven questions, answers were scored using a Likert-type scale from 1 to 4 (Likert, 1932). Survey length, questions suitability, and non-ambiguity of the definitions were considered before survey administration.\n\nData were exported in an Excel file (Microsoft Corp., Redmond, WA, USA) and analyzed by STATA16 (Version 16.1 for Mac, Stata Corp., College Station, TX, USA). Descriptive statistics included mean and standard deviations (SD) or absolute and relative frequencies. We calculated full-scale internal consistency (Cronbach's alpha coefficient) for seven items. Two independent authors (VC and AM) analyzed free text responses to determine possible, significant uncertainties aspects of distance learning.\n\n\nResults\n\nLearning material and duration of the lessons - The learning material (i.e., slides) used previously during the frontal lessons required a significant rearrangement for online delivery. Although the program of the course has remained unchanged compared to previous academic years, online learning has needed the introduction of additional slides. It was necessary to include new slides for 11 of the 13 topics as provided for by the program, and the number of slides as a whole increased by 17.62% (Table 1). Regarding the laboratory activity, online teaching indeed represented a challenge that has been, in part, overcome through two types of approaches. Whenever possible, tutorials were proposed for supporting the realization of experiences at home by using materials readily available. When laboratory experience required the use of specific instrumentation and supplies, videos of the experience/instrumentation have been prepared in the laboratory and then edited by inserting comments and explanations. A total of 12 videos were made and shared on the platform (Table 1).\n\nAlthough no exact quantification was made, the online lessons' duration was always higher than scheduled before. For each lesson, which was meant to last for two hours in the timetable, at least half an hour more was provided; considering that there were no breaks during the online lesson, the increase could be regarded as rather ample.\n\nThe survey - 18 students responded to the survey, and 17 of them were included. 88.2% (15/17) of students were female, and 11.8% (2/17) were male. All students attended online lessons during the COVID-19 lockdown. During online lessons, 76.5% (13/17) of students used PC/Tablet for exclusive use, while 23.5% (4/17) of students were using devices shared with other people. The primary type of Internet access was through a flat-rate fixed network or mobile network with unlimited gigabytes (70.6%, 12/17). Streaming lessons were perceived as the most effective kind of remote lessons (88.2%, 15/17). Overall, distance learning has been good for 29.41% (5/17) of students; many students (58.8%, 10/17) found the online lessons useful even if more tiring. 58.8% (10/17) thought that online learning could be an excellent method to be used during regular activity, but only for particular situations; 17.7% (3/17) believed that online learning should be used during the conventional activity as an alternative learning method. 23.5% (4/17) thought that distance learning could be used only in case of emergencies. All results were reported in Table 2.\n\nOf students, 58.8% (15/17) thought that the platform used for distance teaching (Teams) had been usable enough, followed by 35.3% (6/17), who believed that the platform had been entirely usable. 52.9% (9/17) of students held distance learning adequate for self-study. The level of interaction with teachers and other students was thoroughly satisfying for 94.1% (16/17) and 64.7% (11/17), respectively. During the COVID-19 lockdown, communication with teachers was indicated as good enough by 94.1% of students (16/17). For what concern the level of friendliness perceived during online lessons: 29.4% (5/17) and 41.2% of the interviewees thought there was an adequate level of friendliness, similar to that achieved during face-to-face lessons, while 17.7% (3/17) and 11.8% (2/17) had not perceived a high level of friendliness during online lessons. For 58.8% (10/17) of students, the pandemic situation affected learning negatively; 29.4% (5/17) and 11.8% (2/17) did not perceive any correlation between the general sense of anxiety and his/her learning approach. The results were reported in Table 3. Nine students highlighted some problematic aspects of distance learning: the lack of good Internet access and human interactions with teachers and colleagues, and the difficulties in case of technical applications (as microscope use) were the most common issues about distance learning. Internal consistency (Cronbach's alpha) was equal to 0.68. The value alpha was good, given the aim and field of the study.\n\n(R) Reverse Item\n\n\nDiscussion\n\nTo allow the teaching activities to be conducted at an appropriate level, the first hurdle to overcome is the availability of technological supplies and minimal skills to both students and teachers that lack their own resources. Therefore, when evaluating the online teaching experience, it is essential to exclude that technological limits (related to the use of the web or digital tools) or other external issues would affect the results. In the case presented here, no relevant problems have been raised regarding the use of online tools during the whole course since, as expected, students demonstrated to be familiar with digital resources. At all events, the use of a digital environment requiring little or no specific equipment is preferable in order to cater to everyone.\n\nOverall, our results revealed that online learning is considered an excellent method to use only for particular situations or in emergencies. It should be noted that virtual teaching was not foreseen as part of the education course they had chosen, while the students' expectations are different if they decided to enrol in a distance education course from the beginning. Online synchronous lectures have the characteristics of immediacy, low difficulty, and low cost for both teachers and students (Xie & Zhang, 2020). Accordingly, from our survey, it emerged that they are considered to be the most useful type of online lessons.\n\nAlthough the platform used for virtual classes (Teams) was evaluated from enough to entirely usable by most of the students, it is important to highlight some aspects that may be relevant. This platform allows us to see simultaneously up to nine participants on the screen during a video call, so even in a class with a low number of students, like that described here, it is impossible to have an overview of all the students. This restriction forces the teacher to switch through the users' screen during the lesson since the goal is to include, not exclude, any students. In an attempt to get a picture of the whole class, managing the slides/teaching materials may become difficult. Moreover, the interaction is particularly important in the STEM context (Huang et al., 2020; McDavid et al., 2020) as teachers can target the learning skills and allow students to feel connected to each other, thus building a trustful learning environment. Therefore, online teaching management needs to consider the number of students to avoid such limitations compared to face-to-face lessons that allow us to have a direct, at least visual, contact with all the learners.\n\nInterestingly, the students' attempts to interact with the teacher posing questions, were positively affected by the online delivery of the lessons. On average, at the end of a lesson, the number of questions asked was 2-3, while during online teaching, the number per lesson was never less than 5-6. One may suppose that this is related to less successful communication due to distance learning. However, the complexity level of the questions raised during this course suggested that students needed feedback from the teacher more than a clarification of scientific contents. Sometimes, the mainstream environment does not provide an appropriate level of confidence to the more reserved students, and their participation in the academic discussion remains limited; these students would benefit from a different level of communication access. They overcome their restraint during online experience thanks to the ability to manage their image in the video call (i.e., shutting down the video when speaking or using a low-resolution video) or use chat for conversations. On the other hand, the most confident students have not lost their relationship skills.\n\nThe higher duration of the online lessons observed here may be partly due to the increased number of questions posed by students that took some time to answer exhaustively. On the other hand, the students had more free time in the absence of extra-university activities and the lack of limits deriving from logistics (i.e., dependence on fixed hours for public transport, travel times, etc.). The absence of these limits allowed us to dwell on the most relevant aspects by dedicating attention not conditioned by compliance with the timetable (Deci & Ryan, 2000).\n\nFor what concerns the learning material, from our results emerge an increase in the number of the slides needed for online compared to face-to-face lessons. It seems that classroom interaction plays a fundamental role by allowing the teacher to identify any perplexities or the decline in student attention quickly. It was, therefore, necessary to introduce some slides with the dual objective of bringing students' attention back to the right level and reinforcing information during online lessons.\n\nFrom the perspective of the instructional design, managing the instructional process efficiently will ensure effective and retentive learning. The success of the teaching activity in such contexts relies on the teacher's ability to use multiple technologies and tools to reorganize the teaching and learning process (Merrill et al., 1996).\n\nHowever, for all the possibilities that technology brings to education, one major problem remains when facing practical skills learning; to develop a virtual laboratory class for a master's degree course requires specific skills, both scientific and technological. The role of virtual simulations has been explored first in science and engineering education. More recently, the usefulness of the virtual laboratory in comparison with other instructional methods has been investigated in the life sciences undergraduate courses, however leading to conflicting results (Bonde et al., 2014; Dyrberg et al., 2017; Hofstein & Lunetta, 2004). The suitability of tools depends on the educational purpose, and a different approach is required for practical activities.\n\nMaybe the academic community will need to become more involved by providing resources, expertise, and guidance aimed at the development of specific software/tools for laboratory teaching. At the moment, online education has revealed all its limits regarding students' achievement of adequate familiarity with scientific instrumentation, and the research in this area requires further investigations.\n\nAn education program that includes face-to-face and online lectures might represent a possible learning scenario in all situations requiring restrictive measures but also as an independent, alternative learning method. This blended way of learning (Staker & Horn, 2012) may also take advantage of MOOCs (Kaplan & Haenlein, 2016; Moreno-Marcos et al., 2019) as additional resources based on the learner characteristics, contents, and resources required.\n\nSurprisingly, online lessons have somewhat promoted communication between users of the platform. The virtual room allows students to identify themselves as members of a cultural and micro-culture group, and not to endorse the classical model, which classifies teachers and students as separate categories. In the same way, this offered the teacher the opportunity to become a part of the students' group. The unavoidable intrusion into each other's daily realities helped in developing a relaxed atmosphere in discussions. The natural trust that has been generated has favored the development of a highly empathic class atmosphere by also facilitating the discussion of scientific contents.\n\nThese empirical results are in agreement with the research literature on the role of an informal learning environment as a promoter of motivation and contributing factor to the academic success of students in the area of STEM (McDavid et al., 2020; Salmi & Thuneberg, 2019).\n\nIt is important to note two critical limitations of our study. The most relevant relates to the study design and context. This study aimed to point out the outcomes of a sudden switch from traditional face-to-face to online instruction during the COVID-19 outbreak. Still, the research design was not a controlled experiment with an intervention and control group. The other explicit limitation is the number of participants in the survey. However, the present research is proposed as a case study that becomes more prominent when considering the peculiar conditions that occurred during the pandemic. Moreover, although qualitative, the observed results reveal some dynamics of noteworthy relevance regarding online teaching and its future evolution in the STEM area. For this study's purpose, a theoretical-practical course was considered, thus highlighting the pros and cons of online learning in science and the importance of flexibility across education systems.\n\nThe added value of this study over previous ones is that it refines the role of the educational environment in STEM education, disclosing several unexpected outputs.\n\n\nConclusion\n\nOur study's results highlighted the strengths and weaknesses of online teaching in the STEM area that emerged during the closure of higher education institutions and the suspension of face-to-face lessons due to the pandemic. The main challenges that emerged from our study concern the practical activities that emphasize that online teaching cannot merely be a transposition of face-to-face lessons in a virtual context. To elaborate specific online teaching for practical courses choosing the more suitable tools and enhancing mutual interaction requires special attention. Accurate planning and organization are needed when implementing STEM education, and a major role of the academic community is envisaged as the promoter of advanced, effective tools development for laboratory teaching. More investigation on this topic is necessary to address limits about the student's scientific achievements during virtual instruction. Furthermore, promising outcomes arose from this study. They were evident in the learners' motivations and enhanced communication skills leading to reconsider by the way the educational relationship between teacher and student. In our opinion, when applicable, the blended learning would be the more suitable education program for STEM courses.\n\n\nData availability\n\nFigshare: Questions in English and Italian, https://doi.org/10.6084/m9.figshare.13656029 (Brunelli & Macirella, 2021).\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\n\nConsent\n\nBefore starting the survey all student have been informed of methods and goals of this work. In the email along with the link to accede the survey students have been provided with following information:\n\n“Participation in the online questionnaire”\n\nThis questionnaire is aimed exclusively at the collection of data and information for the evaluation of online teaching. It does not in any way replace the questionnaire that the University submits to students of the final assessment of the course. The data collected will be used exclusively for the preparation of scientific work.\n\nTaking part in the survey implies the declaration of consent. Before completing the questionnaire, please carefully read the information below:\n\n1. the online questionnaire's completion is on a voluntary basis and anonymous; the questionnaire has been created using the online tool Google Forms. More information on how Google treats data can be found at this link: https://policies.google.com/privacy/update. These data will not be recorded on other media or devices, nor will any other data deriving from its navigation on the site be recorded.\n\n2. answers are saved by clicking on the button “submit” and you could suspend the questionnaire at any time;\n\n3. the sensitive personal data of all participants (including the IP address) are not tracked. The protocols on which information passes over the network and data is stored is the HTTPS (HyperText Transfer Protocol over SSL) protocol, a variant of the HTTP protocol that uses, in addition to TCP / IP, the SSL (Secure Sockets Layer) layer that encrypts incoming and outgoing data through a mathematical algorithm.\"", "appendix": "Acknowledgments\n\nWe are grateful to our students for sharing their perspectives and time.\n\n\nReferences\n\nBonde MT, Makransky G, Wandall J, et al.: Improving biotech education through gamified laboratory simulations. Nat Biotechnol. 2014; 32(7): 694–697. PubMed Abstract \| Publisher Full Text\n\nBrunelli E, Macirella R: QUESTIONS ENG.xlsx. figshare. Dataset. 2021. http://www.doi.org/10.6084/m9.figshare.13656029\n\nBussis AM, Chittenden EA: Analysis of an Approach to Open Education. New Jersey Educational Testing Service. 1970.\n\nDeci EL, Rya RM: The \"what\" and \"why\" of goal pursuits: Human needs and the self-determination of behavior. Psychological Inquiry. 2000; 11(4): 227–268. Publisher Full Text\n\nDyrberg NR, Treusch AH, Wiegand C: Virtual laboratories in science education: Students' motivation and experiences in two tertiary biology courses. J Biol Educ. 2017; 51(4): 358–374. Publisher Full Text\n\nHofstein A, Lunetta VN: The laboratory in science education: foundations for the twenty-first century. Sci Educ. 2004; 88(1): 28–54. Publisher Full Text\n\nHuang RH, Liu DJ, Tlili A, et al.: Handbook on facilitating flexible learning during educational disruption: the Chinese experience in maintaining undisrupted learning in COVID-19 outbreak. Beijing: Smart Learning Institute of Beijing Normal University. 2020. Reference Source\n\nHuang RH, Spector JM, Yang JF: Educational Technology: a primer for 21st century. Springer Nature Singapore Pte Ltd. 2019. Reference Source\n\nKaplan AM, Haenlein M: Higher education and the digital revolution: About MOOCs, SPOCs, social media, and the Cookie Monster. Bus Horiz. 2016; 59(4): 441–50. Publisher Full Text\n\nLikert R: A technique for the measurement of attitudes. Arch Psychol. 1932; 22(140): 1–55. Reference Source\n\nMcDavid L, Parker LC, Li W, et al.: The effect of an in-school versus after-school delivery on students' social and motivational outcomes in a technology-based physical activity program. Int J STEM Educ. 2020; 7(1): 28. Publisher Full Text\n\nMerrill MD, Drake L, Lacy MJ, et al.: Reclaiming instructional design. Educational Technology. 1996; 36(5): 5–7. Reference Source\n\nMoreno-Marcos PM, Alario-Hoyos C, Muñoz-Merino PJ, et al.: Prediction in MOOCs: A review and future research directions. IEEE Trans Learn Technol. 2019; 12(3): 384–401. Publisher Full Text\n\nRainbow C: Supporting every teacher: using a video conference platform for teaching online. 2020. Accessed June 03 2020. Reference Source\n\nSalmi H, Thuneberg H: The role of self-determination in informal and formal science learning contexts. Learn Environ Res. 2019; 22(1): 43–63. Publisher Full Text\n\nSingh V, Thurman A: How Many Ways Can We Define Online Learning? A Systematic Literature Review of Definitions of Online Learning (1988-2018). Am J Distance Educ. 2019; 33(4): 289–306. Publisher Full Text\n\nStaker H, Horn MB: Classifying K-12 blended learning. Innosight Institute. 2012. Reference Source\n\nXie G, Zhang Y: School of golden touch? A study of school effectiveness in improving student academic performance. Chin J Sociol. 2020; 7(1): 28. Publisher Full Text" }	[ { "id": "79301", "date": "17 Mar 2021", "name": "Alexander H. Treusch", "expertise": [ "Reviewer Expertise Microbiology", "environmental microbiology", "implementation of virtual laboratory simulations into teaching." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study “Exploring the critical points of teaching STEM subjects in the time of COVID 19: the experience of the course \"Microscopy Techniques for Forensic Biology\" by Brunelli and Macirella describes and analyzes the adaptation of a university course from in person to remote as a result of the lockdown under the COVID-19 pandemic.\nThe study is an important contribution to the discussion on how we can bring the still fairly often traditional, offline university education in natural sciences into the e-learning age. Especially courses that include components designed to transfer practical skills are challenging in this regard, a topic that the study tries to address.\nWhile I think the research framework is well explained, it would be useful for a better understanding of the background of the students to include more information on the regular, pre-COVID course proceedings. Are the students usually using e-learning tools? If yes, which? Is there usually a digital exchange of learning materials (slides, assignments, etc.)? How much are instructional videos used? I would expect that students not used to e-learning tools at all will show a different reaction to those that have used them supporting an in-person course before and only are forced to use them exclusively now.\nAnother thing that would be interesting to include (if the data is available) is how the learning goals were reached with moving the course to online teaching. I assume certain goals, especially of the hands-on training type, would be harder to reach in a remote learning setting. In this context, reporting the results of the evaluation (exam?) in comparison to previous years would give an indication of how well this was managed. However, only downstream courses that require the skills taught here and ultimately the students’ ability to fulfill their tasks in a future employment situation will determine if their learning success was affected by the remote teaching.\nWhile the authors mention virtual laboratory simulations in the discussion, I am wondering if there was a conscious decision made to not use them in the course and if yes, why? Simulations are usually considered very well suited for training students on instruments or letting them experience the workflow of a certain method/procedure. Therefore they are widely used in the training for the operation of technical equipment (e.g. planes, large machines), which are fairly similar, if not more complicated applications to the usage of microscopes.\nWhile, in general, I agree with the discussion and conclusions of the authors, I think there is another aspect to it that is not taken into consideration. Being unfamiliar with the Italian university education system, I get the feeling from the description the authors give in the manuscript that there are quite some differences to e.g. the system in Scandinavian countries. This could explain some of the results obtained by this study. For example, the observed promotion of communication by remote teaching and a subsequent build of trust between students and teachers furthering the academic discussions seems to be something coming from a completely different baseline than what I experience. This could indicate the possibility of a different teaching and learning culture in different countries and might be directly or indirectly related to the fact of how much the use of e-learning tools is already embraced. To be able to generalize the findings of this study I feel this is something that needs to be taken into consideration and discussed.\nAlthough I agree with the authors’ statement that e-learning tools have their limits in educations requiring the training of hands-on skills, there already is quite some progress with the emerging use of virtual laboratory simulations. However, similar to the authors I think they can only supplement the practical parts of a course and not completely replace them. Nevertheless, they are useful tools also outside of an emergency situation as what we have experienced the last year.\nOverall, I think this is a great contribution to the ongoing discussion on implementing e-learning tools into STEM educations. It could benefit from some additional clarifications to better put the study into perspective for people stemming from different university systems.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6479", "date": "12 Apr 2021", "name": "Elvira Brunelli", "role": "Author Response", "response": "The study “Exploring the critical points of teaching STEM subjects in the time of COVID 19: the experience of the course \"Microscopy Techniques for Forensic Biology\" by Brunelli and Macirella describes and analyzes the adaptation of a university course from in person to remote as a result of the lockdown under the COVID-19 pandemic. The study is an important contribution to the discussion on how we can bring the still fairly often traditional, offline university education in natural sciences into the e-learning age. Especially courses that include components designed to transfer practical skills are challenging in this regard, a topic that the study tries to address. While I think the research framework is well explained, it would be useful for a better understanding of the background of the students to include more information on the regular, pre-COVID course proceedings. Are the students usually using e-learning tools? If yes, which? Is there usually a digital exchange of learning materials (slides, assignments, etc.)? How much are instructional videos used? I would expect that students not used to e-learning tools at all will show a different reaction to those that have used them supporting an in-person course before and only are forced to use them exclusively now. Thank you for the suggestions. I accepted and I inserted in the Introduction section a sentence to better clarify the background of students. Despite the good level of confidence with digital tools (developed in different, non-educational contexts), students used few digital resources during the regular pre-COVID courses. In general, the slides showed during the lessons are provided, but both the books and the assignments are of traditional type; moreover, instructional videos are not employed, and the laboratory experiences are delivered during practical laboratory sessions. Another thing that would be interesting to include (if the data is available) is how the learning goals were reached with moving the course to online teaching. I assume certain goals, especially of the hands-on training type, would be harder to reach in a remote learning setting. In this context, reporting the results of the evaluation (exam?) in comparison to previous years would give an indication of how well this was managed. However, only downstream courses that require the skills taught here and ultimately the students’ ability to fulfill their tasks in a future employment situation will determine if their learning success was affected by the remote teaching. For what concerns the examination, the outcome was not different from that of previous cohorts, and all students passed the exams in the ordinary academic session. Instead, I noticed an increase in the internship request for the preparation of the final dissertation. I did not include these data in the paper because it would be better clarified if it was a stochastic event or determined by a curiosity about microscopy developed during the course and not entirely satisfied. I agree that the practical consequences of such online experiences on student formation are delayed, and we cannot at the moment establish if all the students acquired the appropriate practical microscopic skill. While the authors mention virtual laboratory simulations in the discussion, I am wondering if there was a conscious decision made to not use them in the course and if yes, why? Simulations are usually considered very well suited for training students on instruments or letting them experience the workflow of a certain method/procedure. Therefore they are widely used in the training for the operation of technical equipment (e.g. planes, large machines), which are fairly similar, if not more complicated applications to the usage of microscopes. During the course last year, I did not use virtual laboratory since we were absolutely unprepared: the course started in March together with the first lockdown. However, thanks to such experience, I am using a virtual laboratory now, since in Italy we are still in a serious pandemic situation and lessons are mainly delivered online. I hope to merge both methods this year since I am convinced of the utility of simulations to make the student mindful about the methods applied and skills required. While, in general, I agree with the discussion and conclusions of the authors, I think there is another aspect to it that is not taken into consideration. Being unfamiliar with the Italian university education system, I get the feeling from the description the authors give in the manuscript that there are quite some differences to e.g. the system in Scandinavian countries. This could explain some of the results obtained by this study. For example, the observed promotion of communication by remote teaching and a subsequent build of trust between students and teachers furthering the academic discussions seems to be something coming from a completely different baseline than what I experience. This could indicate the possibility of a different teaching and learning culture in different countries and might be directly or indirectly related to the fact of how much the use of e-learning tools is already embraced. To be able to generalize the findings of this study I feel this is something that needs to be taken into consideration and discussed. I agree. This would be an interesting topic to be investigated. I inserted in the text some sentences to introduce these concepts (highlighted). I also suggest that emotional reaction to the worrying pandemic situation may have been relevant in determining the students' reaction. However, this is out of my competencies and expertise. Although I agree with the authors’ statement that e-learning tools have their limits in educations requiring the training of hands-on skills, there already is quite some progress with the emerging use of virtual laboratory simulations. However, similar to the authors I think they can only supplement the practical parts of a course and not completely replace them. Nevertheless, they are useful tools also outside of an emergency situation as what we have experienced the last year. Overall, I think this is a great contribution to the ongoing discussion on implementing e-learning tools into STEM educations. It could benefit from some additional clarifications to better put the study into perspective for people stemming from different university systems." } ] } ]	1	https://f1000research.com/articles/10-89
https://f1000research.com/articles/9-654/v1	29 Jun 20	{ "type": "Research Article", "title": "Bibliometric assessment and implications for practice of sporotrichosis research (1945-2018)", "authors": [ "Priscila Costa Albuquerque", "Bruna de Paula Fonseca e Fonseca", "Fabio Zicker", "Rosely Maria Zancopé-Oliveira", "Rodrigo Almeida-Paes", "Priscila Costa Albuquerque", "Bruna de Paula Fonseca e Fonseca", "Fabio Zicker", "Rosely Maria Zancopé-Oliveira" ], "abstract": "Background: Sporotrichosis has recently emerged as an important mycosis worldwide, with diverse transmission and epidemiologic profiles. For instance, in Brazil most cases are related to zoonotic transmission from naturally infected cats, and the majority of cases in China are due to external injury with environmental materials. Publications on sporotrichosis and on its etiologic agent may guide the direction of the research in this field. It can also define priorities for future studies. Methods: In this study, we evaluated the trends of global research in Sporothrix and sporotrichosis, based on publications records retrieved from Scopus and Web of Science databases for the period of 1945 to 2018. The overall productivity in the field, its geographical and temporal distribution, research themes, co-authorship networks, funding sources, and the implications of research findings for health practice were assessed using bibliometric approaches. Results: A total of 4,007 unique publications involving 99 countries were retrieved, most of them published after 2000. Authors based on institutions from the United States of America and Brazil accounted for 57.4% of the publications. Brazil was the leading country in terms of research collaboration and networking, with co-authorship with 45 countries. The thematic mapping revealed a temporal shift from clinical to applied research. Despite the large number of countries publishing in this field, most of funded studies came from Brazil, Mexico, China, South Africa, or the United States of America. The analysis of content identified few specific public health recommendations for prevention, case-management, or research. Moreover, most papers do not have a clearly defined intended audience. Conclusion: As the research in this field is emerging in several countries, with the generation of a large amount of data, it is necessary that scientists strengthen efforts to translate the research results into practice to curb this neglected infection.", "keywords": [ "Bibliometrics", "Network", "Scientometry", "Sporothrix", "Sporotrichosis" ], "content": "Introduction\n\nSporotrichosis is a subcutaneous mycotic infection caused by dimorphic species of fungi belonging to the genus Sporothrix1. It has a worldwide distribution, a broad range of clinical presentations, and can be fatal as an opportunistic infection in immunosuppressed patients2. For more than one century, the etiological agent of sporotrichosis was identified as the sole species Sporothrix schenckii3. However, in last years, using molecular biology techniques, it was possible to identify other sibling species that also cause sporotrichosis: Sporothrix brasiliensis, Sporothrix globosa, and, to a lesser extent, Sporothrix luriei, Sporothrix pallida, Sporothrix mexicana, and Sporothrix chilensis4–7. These agents can be found in the environment1 and they present different clinical manifestations8, virulence9, drug susceptibility10, and phenotypic characteristics11.\n\nThe research about fungal diseases has been relatively neglected worldwide by public health authorities. The initiative “Global Action Fund for Fungal Infections” (GAFFI), an international non-governmental organization dedicated to combating fungal disease, was created to highlight gaps in diagnostics and treatments for fungal diseases as well as to fund raise and lobby global health agencies12. GAFFI has identified some fungal infections as its highest priorities and sporotrichosis, along with other deep mycoses, is included13. This organization has claimed that sporotrichosis, paracoccidioidomycosis, and fungal keratitis should be included in the WHO’s Neglected Tropical Diseases portfolio. This would be a big step towards increasing research funding and better care for patients with these serious diseases.\n\nBibliometric studies are frequently used to describe the global dynamics of knowledge generation and provide useful information on research discoveries, pointing at the strengths and weakness of new findings14. Through bibliometric analyses, based on quantitative and qualitative indicators, it is possible to assess progress and collaboration in science, technology and innovation15. For instance, the co-authorship of scientific publications reveals collaborative patterns between individuals, organizations, or countries16 and represents a formal statement of interaction between two or more researchers, being widely used to understand and assess collaboration profiles17,18. Bibliometric methods, however, have rarely been applied to the mycology field and not much is known about the extension and trends of Sporothrix research. Therefore, an assessment of the characteristics of the research focusing on sporotrichosis and its etiological agents is necessary to evaluate the progress of the findings in this field and the implications and impacts of sporotrichosis research for health practice.\n\nDriven by the continued expansion of sporotrichosis in some countries such as Brazil, China, Mexico, and India1, we analyzed the global scientific publications and scientific collaboration on the Sporothrix and sporotrichosis research field, with emphasis on the impact of the zoonotic epidemic of sporotrichosis in Brazil2 and on the discovery of the new species genetically related to S. schenckii5. We combined bibliometrics and social network analysis to generate evidence of the dynamics of the research community. Also, implications for practice of the publications were assessed, to check whether the research results were being translated into actions to curb sporotrichosis.\n\n\nMethods\n\nPublications were retrieved from the Web of Science (WoS) and Scopus databases searching for the terms “Sporothrix” or “sporotrichosis” on the title, abstract, and keyword fields. The review included all scientific publications records from 1945 to 2018 (WoS Core Collection). For comparison purposes, the number of publications about other mycoses caused by dimorphic fungi (paracoccidioidomycosis, histoplasmosis, coccidiocomycosis, and blastomycosis) was also obtained, using the genus of the fungus and the name of the mycosis as the search query terms.\n\nThe data retrieved was imported into the text mining software VantagePoint 10.0 (Search Technology Inc. Norcross, GA, USA) and duplicate records were excluded. Names of institutions, countries, funding organizations, and journals were standardized using the VantagePoint list cleanup tool, and further manual processing. An open-access alternative for the use of VantagePoint 10.0 is the software OpenRefine 3.3.\n\nAfter cleaning and standardization, the data was formatted into adjacency matrixes to map co-authorship between countries and institutions based on authors’ professional affiliations. The matrixes were imported into the open-source software Gephi 0.9.1 for network visualization and calculation of metrics19. Degree centrality was used to identify the most central institutions in the network, reflecting the significance of a network member (node) relative to all other nodes in the network. This metric takes into account the diverse means in which a node interacts and communicates with the rest of the network. The most important, or central ones, have a strategic impact in the network. The degree centrality can be explained as the number of direct links that a node has with other nodes. The more relational ties a node has, more power or prestige it may present in a network20. Betweenness centrality was used to recognize organizations that mediated the connection between other institutions and their capacity to control the flow of information in the network20. This metric reveals the extent to which a node works as a bridge among the other nodes in the network, which would otherwise be disconnected. For the spatial visualization of international collaboration, the authors’ professional affiliation country was manually geocoded and processed using the “GeoLayout” 0.9.1.2 and “Map of Countries” 1.5.1 plugins available within Gephi. In these networks, nodes represent countries or an institution, and two or more countries/institutions were connected if their researchers shared the authorship of one or more papers. As co-authorship involves reciprocal collaboration, all connections were considered as non-directional.\n\nTerm maps were created using the VOSviewer 1.6.6 software (Leiden University, the Netherlands) using terms obtained from titles and abstracts of all publications in the database. Each term was graphically denoted by a circle whose diameter and label size were directly proportional to their frequency. The software positions the circles closer to each other according to the power of the relationship and co-occurrence between terms. The mapping allowed a cluster analysis by research themes using a weighted and parameterized variant of modularity-based clustering to recognize groups of correlated terms21.\n\nFunding acknowledgments on publications were only available as a searchable field in WoS and Scopus from late 2008. In order to achieve reliable coverage, only papers published from 2012 onwards were selected for this purpose. Funding agencies were identified, their names standardized (whenever possible), and the number of publications per funding agency summarized.\n\nAbstracts of the publications were reviewed to identify statements or recommendations for sporotrichosis research, case-management, and prevention. Titles and abstracts were tabulated on a Microsoft Excel 2010 spreadsheet, and searched for terms related to statements and recommendations for research, practice or public health. A set of words related to tentative language (“may”, “might”, “speculate”, “suggest”, or “potentially”), prescriptive language (“must”, “propose”, “should”, “stress”, or “recommend”) or related to minimal advice (“consider”, “advise”, “notify”, or “inform”) was used in this process22. The audiences to whom the recommendations were directed to (medical doctors, nurses, laboratory staff, or veterinarians) were also identified.\n\nThe software GraphPad Prism 5 was used to build linear regressions, to check the frequency of publications over time and to compare slopes of different best-fit lines. The Chi-square test was used to test for differences in proportions of “tentative”, “prescriptive”, and “minimal advice” from research results according to the country of origin in an attempt to identify how explicit was the research message to the scientific community, practitioners and public health professionals. A p<0.05 was considered to be significant.\n\n\nResults\n\nSporotrichosis was found to be the mycosis caused by dimorphic fungi with the lowest number of publications in both databases used in this work (Table 1). The literature search on the Sporothrix/sporotrichosis field retrieved 1,868 publications from the WoS database and 3,660 publications from the Scopus database for the period of 1945 to 2018. After removing duplicate references present on both databases, new totals were 1,866 and 3,599 papers, respectively, with 1,458 publications present in both databases. The final data set resulted in 4,007 unique publications (Figure 1; Underlying data23). As depicted in Figure 2a, the overall number of publications on Sporothrix/sporotrichosis has increased steadily over the years. The frequency of publications reflecting the interest in the studied subject varied over time. Four periods could be identified: 1945 to 1962 (Figure 2b), with an average of 10.8±3.7 publications per year; 1963 to 1991 (Figure 2c), 47.2±11.3 papers per year; 1992 to 2002 (Figure 2d), with 63.1±10.3 publications per year; and in the final period, 2003 to 2018 (Figure 2e), 108.9±20.3 papers per year. In two of these periods, 1945–1962 and 1992–2002, the amount of publications per year was approximately constant (slopes of 0.2157 ± 0.1646 and -0.1364 ± 0.9980, p values of 0.2085 and 0.8943, respectively), whereas the two remaining periods, 1963–1991 and 2003–2018, presented increases in the publication numbers per year (slopes of 0.7113 ± 0.2095 and 3.494 ± 0.6573, p values of 0.0021 and 0.0001, respectively). The differences between the slopes were found to be extremely significant (p < 0.0001).\n\n(a) Overall publication numbers per year (1945–2018). Trends in the number of publications on the Sporothrix/sporotrichosis field are represented in a linear regression form for the following time periods: (b) 1945–1962; (c) 1963–1991; (d) 1992–2002; (e) 2003–2018. The continuous line represents the best-fit line for the linear regression. Dashed lines represent the 95% confidence interval of the best-fit line.\n\nAuthors from 99 countries accounted for the 4,007 publications on the Sporothrix/sporotrichosis field. Table 2 lists the 20 most productive countries during the entire period studied and Figure 3 depicts the annual trends of the top 15 countries. The United States of America (USA) and Brazil were the leading countries, publishing together 2,300 publications (57.4% of all publications). The frequency of publications by these two countries differed considerably. After 1973, the USA showed a regular pattern of publications with a range of 15–44 and a median of 25 papers per year, while Brazil had up to 10 publications per year until 1999, with an exponential increase in the number of publications noticed after 2000. Overall, the frequency of publications from the Japan, France, and Canada remained stable throughout the studied period, whereas a significant increase was also observed for Mexico from 2007 onwards, and China from 2009 onwards.\n\nThe diameter of each circle is directly proportional to the annual number of publications. USA: United States of America; BRA: Brazil; JPN: Japan; IND: India; MEX: Mexico; CHN: China; SAF: South Africa; FRA: France; SPN: Spain; CAN: Canada; GER: Germany; NTL: The Netherlands; GBR: Great Britain; ITA: Italy; AUS: Australia.\n\nTable 3 lists the top 15 journals (out of 1,182) with the highest number of articles on Sporothrix/sporotrichosis, along with their respective impact factors. Overall, the journals with most publications were Mycopathologia, Medical Mycology, Mycoses, the International Journal of Dermatology, and JAMA Dermatology. Since Brazil is the current leader on annual publications in this field (Figure 3), the journals harboring these publications were also evaluated separately. When only publications with at least one Brazilian author were considered, in addition to Mycopathologia, Medical Mycology, Mycoses, and the International Journal of Dermatology, the journals PLoS Neglected Tropical Diseases, Frontiers in Microbiology, and four Brazilian scientific journals also appear listed among the top ten journals.\n\nJIF – Journal Impact Factor\n\nN/A – Not available\n\nThe analysis of term maps revealed five broad knowledge areas of the Sporothrix/sporotrichosis research (Figure 4a): description of clinical aspects (green), treatment (yellow), epidemiology and taxonomy (blue), cellular biology (red), and susceptibility assays (pink). The most frequent topics throughout the period studied are represented in a heat map format (Figure 4b), which shows that terms about clinical aspects were more frequent, followed by terms on taxonomy and cellular biology. The analysis in two time periods - 1945–1999 (Figure 4c) and 2000–2018 (Figure 4d) revealed, in this last period of highly increasing publication numbers, a shift from research focused on treatment to other knowledge areas such as epidemiology and taxonomy.\n\n(a) Terms extracted from titles and abstracts clustered into five major research areas: cellular biology, epidemiology and taxonomy, clinical aspects, treatment, and susceptibility assays. Colors indicate clusters of terms that have co-occurred more frequently in the dataset. (b) The heat map shows the most frequent terms in the period analyzed. The frequency was graded from blue to red; where red indicates a higher frequency. (c) Research trends from 1945 to 1999. (d) Research trends from 2000 to 2018. The diameters of the circles on a, c, and d panels are directly proportional to the occurrence of each term. Lines between different circles represent relationships between terms. The colors in c and d thematic maps indicate the occurrence of a term in each period. Blue represents a low occurrence, green an average occurrence, and red a high occurrence.\n\nThe international research network on Sporothrix and sporotrichosis was mapped according to the country affiliation of all authors (Figure 5a). The network is formed by 99 countries, with a Brazilian leadership on collaborations. In fact, Brazilian authors have co-authorship with authors from 45 of the countries that compose the network (Figure 5b). The most frequent Brazilian collaborations were with institutions from the USA (84 publications), followed by the Netherlands (44 publications), Mexico (31 publications), Spain (23 publications), and France (16 publications).\n\n(a) Global research network among 99 countries publishing in the Sporothrix/sporotrichosis field from 1945–2018. (b) Brazilian network of scientific collaborations on the period studied. Country links were mapped based on the authors’ affiliations. Each node represents one country and two countries were considered connected if their researchers shared the authorship of a paper. Only relationships between the first author and their co-authors are shown. Links are color-coded according to the continent of the first author: North America – blue; Africa – dark green; Europe – yellow; South America – light green; Asia – red; Oceania – pink.\n\nTo further understand the dynamics of the Sporothrix/sporotrichosis research in the current leader country of publications, the evolution of Sporothrix research networks in Brazil was analyzed in the two-time intervals, 1945–1999 and 2000–2018, that is, before and after the beginning of the Brazilian zoonotic endemic of sporotrichosis (Figure 6). In total, 29 institutions, mostly from the Southeast region, were present in the first period. The network core (giant component) was formed by 16 institutions: nine universities, one public foundation, and one private laboratory, all from the Southeast region, two universities and one hospital from the South region, one university from the Midwest region and one national research center. The network degree average was 1.5, indicating low connectivity in the network (Figure 6a). The first three institutions with higher degree centrality in that period were federal universities located in the Brazilian Southeast region (Table 4). For the second period, the number of institutions increased almost six times. The network was composed by 169 institutions, 135 of them within the giant component. The degree average increased four times (degree average: 4.426), showing a gain in the network connectivity (Figure 6b). A national research center appears as the most central institution in the network, followed by three federal universities, central actors in the sporotrichosis network throughout the studied period. The ten most central organizations in these networks are shown in Table 4. Notably, most of them are from the Southeast region of the country (70% and 60% for 1945–1999 and 2000–2018, respectively).\n\n(a) Research network from 1945 to 1999. (b) Research network from 2000 to 2018. Each node represents one institution and two institutions were considered connected if their members shared the authorship of a paper. Nodes are color coded according to the geographic location of the institutions: North region - green; Northeast region – orange; Center-West region– yellow; Southeast region- blue; and South region – lilac. Multicampus National institutions are colored in gray. The size of the nodes is proportional to their centrality degree. For visualization purposes, only the giant component is shown. The top ten Brazilian organizations with highest degree centrality are labeled in each panel.\n\nUFRJ: Universidade Federal do Rio de Janeiro, Southeast Brazil; USP: Universidade de São Paulo, Southeast Brazil; UNIFESP: Universidade Federal de São Paulo, Southeast Brazil; UNESP: Universidade Estadual Paulista, Southeast Brazil; UFSM: Universidade Federal de Santa Maria, South Brazil; UERJ: Universidade do Estado do Rio de Janeiro, Southeast Brazil; UFF: Universidade Federal Fluminense, Southeast Brazil; UFMG: Universidade Federal de Minas Gerais, Southeast Brazil; UNB: Universidade de Brasília, Center Western Brazil; UFRGS: Universidade Federal do Rio Grande do Sul, South Brazil; UFPR: Universidade Federal do Paraná, South Brazil; Fiocruz: Fundação Oswaldo Cruz, Brazil.\n\nThe analysis of funding acknowledgments was used as proxy information for research funding. A total of 457 (34%) articles out of 1,310 publications acknowledged funding during the period 2012 to 2018. Table 5 shows the top 10 funding agencies and the number of publications out of a total 236 funding organizations and initiatives. Five of them are from Brazil, two from Mexico, one from China, one from the USA, and one from South Africa.\n\nAfter excluding 1,262 articles with no published abstract, the type of language and the intended audience of the publications were assessed for 2,745 articles (Table 6). Since Brazil was the current leading country publishing in the Sporothrix/sporotrichosis field, we compared the frequency of statements related to research and practice on papers with at least one Brazilian author with those of authors from other countries exclusively. The proportion of papers presenting language compatible with tentative (p = 0.0003) or minimal advice (p = 0.0139) statements for sporotrichosis research was higher in the papers co-authored by Brazilians. The specific audience for the statements was mentioned in 167 abstracts. As depicted in Figure 7, most of the implications for practice were directed to medical doctors, followed by veterinarians, nurses, and laboratory technicians. Only 16 publications presented more than one audience, as indicated at the abstract. Some examples of actionable messages and their audiences are listed in Table 7.\n\nThe Venn diagram was constructed based on four different audiences observed in the abstracts of the publications studied. Numbers on diagrams indicate how many abstracts reporting advice for medical doctors, veterinary doctors, nurses, and/or lab technicians were identified.\n\n\nDiscussion\n\nThe results herein presented demonstrate that research on Sporothrix and sporotrichosis is increasing worldwide. This increase could be even greater, because some regional or specialized journals may possibly not be indexed in the databases reviewed. However, considering the proposed focus, the authors believe that the study material was a comprehensive representation of the scientific production in the field.\n\nThe apparent fast engagement of authors from several countries in the late 90s can be explained by the global increase in the incidence of sporotrichosis1, especially in Brazil2 and China24, two of the major countries currently publishing on this field. Historically, Brazil and China are also the leading countries reporting sporotrichosis cases, followed by South Africa, all of them with more than 3,000 human and animal cases reported25. In this study, South Africa ranked as the seventh country on number of publications with no increasing trend. Most of the cases in this country occurred during an outbreak in the 1940’s26. Although a new mine-related outbreak occurred more recently27, there is no evidence that there is a re-emergence of sporotrichosis occurring in South Africa, which may explain its apparently constant number of publications in this field.\n\nThe number of publications on the Sporothrix/sporotrichosis field usually followed crucial events that have driven to a shift on typical clinical cases of sporotrichosis. In the 1980s, with the development of ketoconazole, the first oral antifungal azole drug28, and the emergence of AIDS, there was an increase in papers reporting results of sporotrichosis treatment29,30 and unusual severe forms of sporotrichosis related to immunosuppression31–33, coherent with the predominant thematic at the time. After 2007, the advances in polyphasic taxonomy of Sporothrix spp., driven by the worldwide increasing numbers of clinical cases5, resulted in the description of several new pathogenic Sporothrix species5–7, which may explain the shift for epidemiology and taxonomy occurred in this century.\n\nAuthors from Brazil and the USA authored around 57% of the papers on the subject studied. Also, a high-level of collaboration between these two countries was seen, as occurred in other knowledge areas34,35. While the number of publications authored by researchers from the USA showed a consistent trend in the studied period, Brazilian authors have emerged as very productive in this field. Some factors that may have influenced the strong commitment of Brazilian researchers in the Sporothrix/sporotrichosis field include: (i) the zoonotic sporotrichosis epidemic, that begun in Rio de Janeiro state in 200036 and now is spreading to almost the totality of the Brazilian territory37–39; (ii) the increasing numbers of national and international Brazilian collaboration networks40; and (iii) the beginning and still discrete recognition of the relevance of this research field by Brazilian funding agencies, which was detected in the funding analysis of this study.\n\nThe evaluation of co-authorship networks in sporotrichosis identified structural patterns of research involving Brazilian scientists. USA, Netherlands, Mexico, Spain, and France were Brazil’s most frequent collaborators. Government collaboration and research programs supported by the USA are in place since 1950’s, explaining the robust scientific collaboration between the countries34.\n\nThe Brazilian sporotrichosis research network has grown, almost six times in size, from the first (initial 54 years of low productivity) to the second period (last 18 years with a high level of publications), over the seven decades evaluated. This fact, together with the increase in the average degree and size of the giant component, indicated a strengthening of network cohesion for collaboration over the years. It is worth noting that the measure of centrality is not related to the volume of publications, but to the capacity to aggregate collaboration. The centrality analysis of the interinstitutional network highlighted the current role of one national research center in promoting collaboration and knowledge spreading in the sporotrichosis field. This same research center also has a centrality on the research of other infectious diseases15,41. High degree centrality from a research center indicated a strong collaborative pattern in research15. Together with other institutions, the Federal Universities in Southeast Brazil had a vital role in maintaining the connection between the overall research network and in ensuring that the less well-connected organizations gained access to new knowledge and information on sporotrichosis during the period studied. It is interesting to note that just recently, sporotrichosis has spread to the Northeast39 and Center-Western regions of the country38, which means that, in the future, institutions from these regions may have a more important role in the collaboration network.\n\nThe network evolution was accompanied by a shift in the research trends. When comparing the first period to the 2000 onwards, the basic biomedical research profile gained more importance and became most frequent. The diversity of research trends may be related to the continued increase of institutions engagement in the Sporothrix/sporotrichosis network, providing new insights through new collaborations, showing the effectiveness of the research network in knowledge generation, sharing and diffusion. A similar scenario was observed in dengue research networks42.\n\nThe current funding scenario for fungal infections has a negative perspective. Only cryptococcal meningitis was classified within the most poorly funded neglected diseases by the G-Finder survey (a reference publication for research funding flows on neglected infectious), receiving less than 0.5% of global funding43. Other clinically relevant fungal infections (paracoccidioidomycosis, mycetoma, sporotrichosis, and chromoblastomycosis) were not even quoted in the G-Finder report. In fact, a recent study noticed that some fungal diseases, including sporotrichosis, have received negligible funding44. Our study corroborates these findings, showing that, with the exception of Brazil, possibly because of the expanding sporotrichosis endemic areas37–39, other countries are not strongly committed to sporotrichosis funding research.\n\nIn our analysis, publications co-authored by Brazilians researchers have more tentative and minimal advice or recommendations for practice than papers written by authors from other countries. This could be related to a concern of Brazilian researchers to address the major public health problem that sporotrichosis has become in the country2,37, which has certainly impacted the overall number of publications. The review based solely on abstracts is a limitation of this study. The fact that some journals have a specific audience (for instance, four of the top ten journals are directed to dermatologists) may bias the nature of recommendations. However, as abstracts are the first contact for the readers with a publication, we believe mentioning statements or recommendations for practice in this section can improve the visibility of the research. Also, the analysis of contents revealed that a minor proportion of publications is directed to more than one specific audience, which is one indicator of a poor translational research in the studied field.\n\nDespite cases being reported all around the globe1, sporotrichosis remains a neglected disease in terms of research interest, funding and medical attention. The growth of research on sporotrichosis needs to be translated into practical applications on diagnosis, treatment, and prevention, given the limited tools available for rapid tests2, the cost-effective treatment45, and the lack of effective vaccines46. The challenge is to share and advance knowledge to curb this disease. The funding agencies have a critical role to play in this context.\n\n\nData availability\n\nUnderlying data generated in this study are available at Open Science Framework: Bibliometric assessment of sporotrichosis research. https://doi.org/10.17605/OSF.IO/MXU6V23\n\nThis project contains the following underlying data:\n\n- (F1_T1) Countries publication by years.xlsx (All countries with sporotrichosis publications)\n\n- (F2) MapVoS.txt (Map file for VOSViewer software)\n\n- (F2) NetVoS.txt (Network file for VOSViewer software)\n\n- (F3) CountryNet.gephi (Country map file for Gephi software)\n\n- (F4) Period I.gephi (Network file for Gephi Software)\n\n- (F4) Period II.gephi (Network file for Gephi Software)\n\n- (T2) Source.xlsx (All Journals with retrieved publications about sporotrichosis)\n\n- (T3) Funding BR 12_18.xlsx (Funding agencies supporting sporotrichosis research)\n\n- (T4) Actionable messages.xlsx (implications for practice on retrieved articles)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nChakrabarti A, Bonifaz A, Gutierrez-Galhardo MC, et al.: Global epidemiology of sporotrichosis. 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PubMed Abstract \| Publisher Full Text\n\nGovender NP, Maphanga TG, Zulu TG, et al.: An outbreak of lymphocutaneous sporotrichosis among mine-workers in South Africa. PLoS Negl Trop Dis. 2015; 9(9): e0004096. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGupta AK, Lyons DCA: The rise and fall of oral ketoconazole. J Cutan Med Surg. 2015; 19(4): 352–7. PubMed Abstract \| Publisher Full Text\n\nMartins JE, Mendonça PR, Cucé LC: Tratamento da esporotricose com cetoconazol. Rev Hosp Clin. 1982; 37(2): 92–4.\n\nSamorodin CS, Sina B: Ketoconazole-treated sporotrichosis in a veterinarian. Cutis. 1984; 33(5): 487–8. PubMed Abstract\n\nFitzpatrick JE, Eubanks S: Acquired immunodeficiency syndrome presenting as disseminated cutaneous sporotrichosis. Int J Dermatol. 1988; 27(6): 406–7. 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Mem Inst Oswaldo Cruz. 2001; 96(6): 777–9. PubMed Abstract \| Publisher Full Text\n\nGutierrez-Galhardo MC, Freitas DFS, do Valle ACF, et al.: Epidemiological aspects of sporotrichosis epidemic in Brazil. Curr Fungal Infect Rep. 2015; 9(4): 238–45. Publisher Full Text\n\nAraújo LTR, Silva WA, Juliano RS: Região fronteriça e epidemiologia: Estudo da esporotricose e sua relação na dinâmica da fronteira Brasil-Bolívia. Rev GeoPantanal. 2017; 12: 97–105. Reference Source\n\nSilva GM, Howes JCF, Leal CAS, et al.: Surto de esporotricose felina na região metropolitana do Recife. Pesq Vet Bras 2018; 38(9): 1767–71. Publisher Full Text\n\nLang PB, Gouveia FC, Leta J: Health research networks on the web: an analysis of the Brazilian presence. Cad Saude Publica. 2014; 30(2): 369–78. PubMed Abstract \| Publisher Full Text\n\nMota FB, Fonseca BPFE, Galina AC, et al.: Mapping the dengue scientific landscape worldwide: a bibliometric and network analysis. Mem Inst Oswaldo Cruz. 2017; 112(5): 354–63. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFonseca BPFE, Zicker F: Dengue research networks: building evidence for policy and planning in Brazil. Health Res Policy Syst. 2016; 14(1): 80. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChapman DN, Doubell A, Oversteegen L, et al.: Policy cures research. 121.\n\nRodrigues ML, Albuquerque PC: Searching for a change: The need for increased support for public health and research on fungal diseases. PLoS Negl Trop Dis. 2018; 12(6): e0006479. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMahajan VK: Sporotrichosis: An Overview and Therapeutic Options. Dermatol Res Pract. 2014; 2014: 272376. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGarcía-Carnero LC, Pérez-García LA, Martínez-Álvarez JA, et al.: Current trends to control fungal pathogens: exploiting our knowledge in the host-pathogen interaction. Infect Drug Resist. 2018; 11: 903–13. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "71160", "date": "24 Sep 2020", "name": "Max Carlos Ramírez-Soto", "expertise": [ "Reviewer Expertise Medical Mycology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nCongratulations to the authors. Despite sporotrichosis remains a neglected disease in terms of research interest, funding and medical attention, to which the authors have made significant contributions. However, this study has several major observations:\nMethods. The literature search is limited to two databases. Fig. 1. Present a flow, and follow the guidelines of a scoping review. MedLine / Pubmed and Abstracts of Scientific Conference should be reviewed. There are several published studies on sporotrichosis in journals indexed in SCIELO, LiLACS, etc. These papers have not been included in the study.\nI don’t think that this article contains enough robust data to evidence the implications for practice. The strategy and organization of the information about Implications for practice is not appropriate. A set of words related to tentative language is biased. Furthermore, only the abstracts have been reviewed. To evaluate the implications in practice, it is necessary to organize the different types of published studies: case series, observational study, clinical trials and systematic review, diagnostic test, etc. Furthermore, studies not related to the clinical practice of sporotrichosis should be excluded.\nResults: Paragraph 1. I suggest include in Results section include the statistical test. Scientific production by hyperendemic areas of sporotrichosis should be presented.\nTable 3. I suggest to make a comparison between Brazil, USA, Mexico, China, or hyperendymic areas of sporotrichosis.\nThe authors should include the different types of published articles: review, original, short communication, case reports, clinical trial, systematic, review, correspondece, Research Letters, etc.\nResearch trends. It would be interesting to know how these trends vary between Latin America. Europe and Asia.\nImplications for practice I suggest organizing the published studies: case series, observational study, clinical trials and systematic review, diagnostic test, etc. Although there are very few clinical trials and systematic reviews on sporotrichosis, this evidence should be presented in tables. Furthermore, studies not related to the clinical practice of sporotrichosis should be excluded. Finally, sporotrichosis is not limited to dermatology.\nDiscussion In the “Discussion” section I would have wished to see more information on implicance practic . Limitations should be described and discussed such as: the number of databases included in the study, the studies were not classified, to differentiate the type of scientific evidence for clinical practice, etc.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "6510", "date": "09 Apr 2021", "name": "Rodrigo Almeida-Paes", "role": "Author Response", "response": "We would like to thank this reviewer for his thoughtful comments. Our point-by-point response is presented below: Reviewer comment: Methods. The literature search is limited to two databases. Fig. 1. Present a flow, and follow the guidelines of a scoping review. MedLine / Pubmed and Abstracts of Scientific Conference should be reviewed. There are several published studies on sporotrichosis in journals indexed in SCIELO, LiLACS, etc. These papers have not been included in the study. Our response: The authors used a bibliometric analysis approach to observe the evolution of the world scientific literature and to identify research trends in the sporotrichosis field, focusing on the Brazilian scenario. The authors did not intend to assess the state of the art in the area, but rather to perform a bibliometric analysis with the research effort, which differs from a scoping review. The authors chose as methodological approach not to use regional bases such as SciELO and LILACS, due to the difficulty to obtain total equivalence for a global analysis, since other regional bases should be included, making data harmonization difficult. Scopus includes a more expanded spectrum of journals (covers 100% of Medline and Embase) and Web of Science covers the oldest publications. Also, metadata for multiple affiliations for each author or contributor are included in Pubmed only at the end of 2014. The authors understand the relevance and challenges of integrating different databases in bibliometric analysis to provide a reliable research panorama. The inclusion of limitations will clarify any methodological bias. Action taken: We included a paragraph in the discussion addressing this limitation of study. Reviewer comment: I don’t think that this article contains enough robust data to evidence the implications for practice. The strategy and organization of the information about Implications for practice is not appropriate. A set of words related to tentative language is biased. Furthermore, only the abstracts have been reviewed. To evaluate the implications in practice, it is necessary to organize the different types of published studies: case series, observational study, clinical trials and systematic review, diagnostic test, etc. Furthermore, studies not related to the clinical practice of sporotrichosis should be excluded. Our response and action taken: The text was amended to describe in the methodology the recovery of recommendation messages for practice, research and public health in abstracts based on a wording recovery. Several authors have already demonstrated the importance of abstracts, since they are a condensed version of a scientific research and the most often read section of a research article. Searching abstracts are convenient because they contain most of the article's relevant keywords and appear to be a useful tool to inform to practitioners as a resource for guiding clinical decisions. The tentative set of words was already validated by Lynn J et al 2011, where research articles from three leading clinical journals (N Engl J Med, JAMA, and Ann Intern Med) were examined to identify terms that articulated implications (eg, words such as ‘may’, ‘speculate’, ‘suggest’, ‘reinforce’ and ‘potentially’). Reviewer comment: Paragraph 1. I suggest include in Results section include the statistical test. Our response and action taken: This information was included. Reviewer comment: Table 3. I suggest to make a comparison between Brazil, USA, Mexico, China, or hyperendymic areas of sporotrichosis. Scientific production by hyperendemic areas of sporotrichosis should be presented. Our response and action taken: Table 2 was replaced for an improved version as suggested. To address the reviewer’s suggestion, the authors included the proportion of publications compared to the total number of articles for each country in the table 1, which shows Top 20 most active countries in the Sporothrix/sporotrichosis field according to author’s country of professional affiliation what allows comparison between hyperendemic countries. Reviewer comment: The authors should include the different types of published articles: review, original, short communication, case reports, clinical trial, systematic, review, correspondece, Research Letters, etc. Our response: The sentence in methods was altered in order to clarify the point raised by the reviewer identifying the different types of articles analyzed (original articles, reviews, letters and editorials). Action taken: See Souce of data section. Reviewer comment: Research trends. It would be interesting to know how these trends vary between Latin America. Europe and Asia. Our response: Thank you for this suggestion. The authors agree that this analysis would show a bigger picture. However, for a robust and reproducible analysis, the quantity of publications must be consistent to construct a map based on a co-occurrence matrix. To assess how these trends vary between Latin America, Europe and Asia, the small size of text corpus of each region may not be able, unfortunately, to represent nuances in the research scenario. Reviewer comment: Implications for practice: I suggest organizing the published studies: case series, observational study, clinical trials and systematic review, diagnostic test, etc. Although there are very few clinical trials and systematic reviews on sporotrichosis, this evidence should be presented in tables. Furthermore, studies not related to the clinical practice of sporotrichosis should be excluded. Our response: The text was amended to describe the methodology to recovery recommendations messages for practice, research and public health in abstracts based on wording recovery, rather than a comprehensive search for recommendations for practice. Our original intention with this research was to evaluate a methodology to identify messages and technical recommendations for action in the scientific publications on sporotrichosis. In this first moment, the authors do not intend to analyze the messages, but the validation of the recovery methodology. Further analysis with a broader sample of medical journals to examine how researchers report evidence for action, aiming to assess how they framing their research findings to address implications for practice and public health is needed. Action taken: Changes in the title, abstract (methods section), introduction (last paragraph), results (statements about practice section), and discussion Reviewer comment: Finally, sporotrichosis is not limited to dermatology. Our response and action taken: Thank you for the reminder. The authors were refering to journals with the scopus in dermatology. The text was amended to: “for instance, four of the top ten journals are directed to medical doctors with interest in dermatology”. Reviewer comment: Discussion: In the “Discussion” section I would have wished to see more information on implicance practic. Limitations should be described and discussed such as: the number of databases included in the study, the studies were not classified, to differentiate the type of scientific evidence for clinical practice, etc. Our response and action taken: Thank you for this observation. To clarify the limitations the text was amended and a limitations section was included in the end of the discussion. To address the reviewer’s suggestion, a paragraph describing the applicability of the methodology proposed to the retrieval of the key messages was also included in the discussion section." } ] }, { "id": "77889", "date": "15 Feb 2021", "name": "Maria Simone Menezes Alencar", "expertise": [ "Reviewer Expertise Scientific and technological information acting mainly on the following subjects: bibliometrics", "technological foresight", "patent analysis and open science." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe topic of the work is relevant, and the bibliometric approach is adequate. The content analysis proposal is innovative and deserves to be highlighted. It is strongly recommended that searches in bibliometric studies be conducted in specialized databases. Although the used methodology which includes data from several sources is strong, the study should include other data sources in the field. I also suggest the inclusion of the Medline database in this study. I suggest that only articles published in journals be used, as they are more relevant documents that report completed research. Peer-reviewed journals provide scientific credibility and are not as biased as other indexed documents, such as letters, notes, editorials, etc. Although the methodological stage “Implications for practice” shows an interesting innovation in its approach, it should be interpreted with care. To validate the results, I suggest that this stage be improved with two independent experts analyzing a statistical percentage of the sample. Although the analysis of studies on the subject in the four periods shows different behaviors, these ranges are not used in other analyses. In this way, this presentation is somewhat disconnected with the general context of the article. The other results are well presented and consistently discussed.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6516", "date": "09 Apr 2021", "name": "Rodrigo Almeida-Paes", "role": "Author Response", "response": "We would like to thank this reviewer for her helpful comments that helped to improve the quality of our manuscript. Our point-by-point responses are presented below: Reviewer comment: It is strongly recommended that searches in bibliometric studies be conducted in specialized databases. Although the used methodology which includes data from several sources is strong, the study should include other data sources in the field. I also suggest the inclusion of the Medline database in this study. I suggest that only articles published in journals be used, as they are more relevant documents that report completed research. Peer-reviewed journals provide scientific credibility and are not as biased as other indexed documents, such as letters, notes, editorials, etc. Our response: The authors chose as methodological approach not to use regional bases as SciELO and LILACS, due to the difficulty to obtain total equivalence for a global analysis, since other regional bases should be included and the difficulty of data harmonization. Scopus includes a more expanded spectrum of journals (covers 100% of Medline and Embase) and Web of Science covers the oldest publications. Also, metadata for multiple affiliations for each author or contributor are included in Pubmed only at the end of 2014. Action taken: The sentence in methods was altered in order to clarify the point raised by the reviewer identifying the different types of articles analyzed (original articles, reviews, letters and editorials). Please see Souce of data section. Reviewer comment: Although the methodological stage “Implications for practice” shows an interesting innovation in its approach, it should be interpreted with care. To validate the results, I suggest that this stage be improved with two independent experts analyzing a statistical percentage of the sample. Our response: We appreciate the sugestion. The proposal of this research was to evaluate a methodology to identify messages and technical recommendations for action in the abstracts of scientific publications on sporotrichosis. In this first moment, the authors do not intend to analyze the messages, but the validation of the recovery methodology. And, in fact, two of the authors reviewed the data. In the further analysis, in order to validate the recovery of recommendations, two independently experts will always review the recommendations. Action taken: The text has been modified to clarify that this was a methodological evaluation proposal, rather than a comprehensive analysis of implications for practice. We also clarified that two evaluators collected the data. Reviewer comment: Although the analysis of studies on the subject in the four periods shows different behaviors, these ranges are not used in other analyses. In this way, this presentation is somewhat disconnected with the general context of the article. The other results are well presented and consistently discussed. Our response: The reviewer raised an important point. Our initial analysis of the four periods revealed that two of them, 1945-1962 and 1992-2002, publications remained without significant variation. The strongest variation occurred after 2002, which coincides with the start of publications about the Brazilian zoonotic epidemics of sporotrichosis. Therefore, for subsequent analysis, we chose to separate the articles only in two periods: before and after the beginning of the Brazilian sporotrichosis epidemics. Action taken: The issue raised by the reviewer was clarified in the research trends section of results and further addressed in the discussion." } ] }, { "id": "77886", "date": "24 Feb 2021", "name": "Daniel Zamith-Miranda", "expertise": [ "Reviewer Expertise Microbiology (mycology) and immunology." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript \"Bibliometric assessment and implications for practice of sporotrichosis research (1945-2018)\" from Albuquerque and colleagues shows how research on sporotrichosis has been conducted in the mentioned time range. The authors raised relevant data to conclude where and how this research has been done. Besides usual typos, parts of the text, as well as some figures should be remade (comments below).\nIntroduction When citing reference #15, the authors should highlight that the cited bibliometric analyses were about tuberculosis. Otherwise, the reader may think that those analyses were already made for sporotrichosis and that would decrease the impact of the current work. End of third paragraph: “impact” is more appropriate than “impacts”.\nMethods Is citation #19 correctly referenced? Isn’t the publishing year supposed to be included at least?\nResults What is reference 23? Is it a preprint? Is it the same work as this manuscript published somewhere else? Table 3: Why does the Rank of all publications, after going from 1 – 10, go to 22, 24, 23, 21, and 26? Isn’t that supposed to be the top 15 journals? Figure 4: From A to D, most of what is written is way too small. Even when zooming in, some words are difficult to read. I recommend the authors to at least increase the resolution, but also try to rearrange the panels in order to make it easier for the reader. Even 4c and 4D have distinct resolution, even though the text is supposed to be identical among them. Fig. 4 Legend: “(b) The heat map shows the most frequent terms in the period analyzed”. Please specify the period. Fig 5A: I am not sure if this figure is adding information or even helping the understanding of the text. The authors should consider to change the format, or even take it out of the manuscript. Fig 6: It seems that, besides the research networks, this figure is also about the distribution of scientific institutions in Brazil. Merging the current figure with a map would make it easier to visualize. Table 7: Consider to keep it as supplementary information.\nDiscussion In “…This fact, together with the increase in the average degree and size of the giant component…”. Please clarify what is the giant component. In “…It is interesting to note that just recently, sporotrichosis has spread to the Northeast and Center-Western regions of the country…”. For the sake of being consistent with the nomenclature used in Results, please change Center-Western to Midwest. In “…In our analysis, publications co-authored by Brazilians researchers have more tentative and minimal…”, change to Brazilian researchers. I agree with the other reviewer that conclusions regarding implications in practice cannot be taken based on the method used.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "6517", "date": "09 Apr 2021", "name": "Rodrigo Almeida-Paes", "role": "Author Response", "response": "The authors thank the careful revision of our manuscript. Our point-by-point response to the reviewers comments are presented below: Reviewer comment: When citing reference #15, the authors should highlight that the cited bibliometric analyses were about tuberculosis. Otherwise, the reader may think that those analyses were already made for sporotrichosis and that would decrease the impact of the current work. End of third paragraph: “impact” is more appropriate than “impacts”. Our response and action taken: Thank you for the corrections. We have added reference #15 to emphasize the potential of network analysis. We have clarified the article findings as suggested. Reviewer comment: Methods: Is citation #19 correctly referenced? Isn’t the publishing year supposed to be included at least? Our response and action taken: Thank you for the corrections. All references were carefully reviewed and corrected as suggested. The correct year is 2009. Reviewer comment: Results: What is reference 23? Is it a preprint? Is it the same work as this manuscript published somewhere else? Our response and action taken: This reference refers to the raw data generated in this study that are available at Open Science Framework: Bibliometric assessment of sporotrichosis research. https://doi.org/10.17605/OSF.IO/MXU6V This was a requirement of the journal. Reviewer comment: Table 3: Why does the Rank of all publications, after going from 1 – 10, go to 22, 24, 23, 21, and 26? Isn’t that supposed to be the top 15 journals? Our response: Our intention with this table is to show the top 10 global journals and the top 10 journals publishing Brazilian research. Sometimes a journal within the Brazilian top 10 is not within the top 10 global journals, PLoS Neglected Tropical Diseases, for instance (top 6 Brazilian and 24th global). Action taken: Table 3 caption was amended to include the description of rank comparison. Reviewer comment: Figure 4: From A to D, most of what is written is way too small. Even when zooming in, some words are difficult to read. I recommend the authors to at least increase the resolution, but also try to rearrange the panels in order to make it easier for the reader. Even 4c and 4D have distinct resolution, even though the text is supposed to be identical among them. Fig. 4 Legend: “(b) The heat map shows the most frequent terms in the period analyzed”. Please specify the period. Our response and action taken: Regarding Figure 4 resolution, we gave our best to increase font letters as requested but, to do it, figure would be larger than the page size. However, all terms that generated the figure are presented in the (F2) NetVoS.txt (Network file for VOSViewer software) underlying data, available at https://doi.org/10.17605/OSF.IO/MXU6V. The legend of the figure was rewritten, to comply with the reviewer's request. Reviewer comment: Fig 5A: I am not sure if this figure is adding information or even helping the understanding of the text. The authors should consider to change the format, or even take it out of the manuscript. Our response and action taken: According to the reviewer suggestion, the figure was removed from the manuscript. Reviewer comment: Fig 6: It seems that, besides the research networks, this figure is also about the distribution of scientific institutions in Brazil. Merging the current figure with a map would make it easier to visualize. Our response: Unfortunately, we were not able to merge this figure with a map. We apologize for this situation. Reviewer comment: Table 7: Consider to keep it as supplementary information. Our response: F1000Research does not accept supplementary material, so we kept the table within the main article. Reviewer comment: In “…This fact, together with the increase in the average degree and size of the giant component…”. Please clarify what is the giant component. Our response and action taken: The giant component is the network core (Mentioned in the results section - Network evolution among Brazilian institutions). This information was added to the text. Reviewer comment: In “…It is interesting to note that just recently, sporotrichosis has spread to the Northeast and Center-Western regions of the country…”. For the sake of being consistent with the nomenclature used in Results, please change Center-Western to Midwest. Our response and action taken: The text was changed accordingly. Reviewer comment: In “…In our analysis, publications co-authored by Brazilians researchers have more tentative and minimal…”, change to Brazilian researchers. Our response and action taken: The text was changed accordingly. Reviewer comment: I agree with the other reviewer that conclusions regarding implications in practice cannot be taken based on the method used. Our response and action taken: As requested by all reviewers, the text has been rewritten to demonstrate the proposal for key message recovery methodology and not to conclude implications for practice." } ] } ]	1	https://f1000research.com/articles/9-654
https://f1000research.com/articles/9-1477/v1	17 Dec 20	{ "type": "Research Article", "title": "Inter-site and interpersonal diversity of salivary and tongue microbiomes, and the effect of oral care tablets", "authors": [ "Hugo Maruyama", "Ayako Masago", "Takayuki Nambu", "Chiho Mashimo", "Kazuya Takahashi", "Toshinori Okinaga", "Ayako Masago", "Takayuki Nambu", "Chiho Mashimo", "Kazuya Takahashi", "Toshinori Okinaga" ], "abstract": "Background: Oral microbiota has been linked to both health and disease. Specifically, tongue-coating microbiota has been implicated in aspiration pneumonia and halitosis. Approaches altering one's oral microbiota have the potential to improve oral health and prevent diseases. Methods: Here, we designed a study that allows simultaneous monitoring of the salivary and tongue microbiomes during an intervention on the oral microbiota. We applied this study design to evaluate the effect of single-day use of oral care tablets on the oral microbiome of 10 healthy individuals. Tablets with or without actinidin, a protease that reduces biofilm formation in vitro, were tested.\nResults: Alpha diversity in the saliva was higher than that on the tongue without the intervention. The core operational taxonomic units (OTUs) common to both sites were identified. The salivary and tongue microbiomes of one individual tended to be more similar to one another than to those of other individuals. The tablets did not affect the alpha or beta diversity of the oral microbiome, nor the abundance of specific bacterial species. Conclusions: While the salivary and tongue microbiomes differ significantly in terms of bacterial composition, they show inter- rather than intra-individual diversity. A one-day usage of oral care tablets did not alter the salivary or tongue microbiomes of healthy adults. Whether the use of oral tablets for a longer period on healthy people or people with greater tongue coating accumulation shifts their oral microbiome needs to be investigated.", "keywords": [ "oral care tablet", "oral microbiome", "actinidin", "QIIME 2" ], "content": "Introduction\n\nOral microbiota is a collection of microorganisms that reside in the oral cavity. It has been linked to the promotion of both health and disease1,2. Among the different tissues in the oral cavity, the tongue is considered a dominant source of oral microbial populations3,4. Further, tongue coating is proposed to cause oral malodor5 or, upon sudden dissociation, aspiration pneumonia in elderly people with impaired defense mechanisms6,7. In addition, the tongue coating is a risk indicator of aspiration pneumonia in edentate individuals8.\n\nA variety of methods to reduce tongue coating have been developed and tested to reduce oral malodor9,10. Mechanical removal of the tongue coating using tongue brushes or tongue cleaners is one such popular method9,11. Other methods include using antimicrobials, e.g., in gels or mouthwashes, or using oral tablets10,12.\n\nThe tongue microbiota in elderly individuals has been classified into several types with characteristic bacterial composition. These types correlate with the risk to aspiration pneumonia4,13. Therefore, methods that could alter the tongue microbiota to a healthy microbiota type could contribute to oral health. We have previously reported that tongue brushing does not alter the alpha or beta diversity of oral microbiota in healthy adults14,15. By contrast, according to a recent study, the use of oral care tablets decreases the amount of volatile sulfur compounds (VSCs) produced by bacteria16. Further, oral care tablets that contain actinidin, a cysteine protease found in kiwifruit, reduce oral biofilm formation in vitro12. However, it is not clear whether these interventions affect the oral microbiota as a whole or the abundance of specific bacteria.\n\nIn the current study, we examined the effect of oral care tablets with and without actinidin on the salivary and tongue microbiomes of healthy individuals. We also investigated the diversity of the salivary and tongue microbiomes, and interpersonal microbiome diversity. We show that alpha diversity of the salivary microbiome was greater than that of the tongue microbiome; that an individual’s salivary and tongue microbiomes were more similar to one another than to those of another individual; and that the oral care tablets did not affect the oral microbiomes in the population tested. These findings add to the knowledge of the interpersonal diversity and dynamics of the oral microbiota in humans.\n\n\nMethods\n\nTen healthy adults participated in the study, with three different treatments tested: two different types of oral tablets (with or without protease), and a negative control (no tablet). For the tablet treatments, the saliva and tongue coating were collected between October 2016 and November 2017 at participants’ home (mainly in Osaka, Japan, and in some cases nearby prefectures). DNA extraction and data analysis were conducted at Department of Bacteriology, Osaka Dental University (Hirakata, Japan).\n\nParticipants were recruited from faculty members and graduate students working at the Osaka Dental University hospital, as well as from dentists who were acquainted with an author of this study. Ten healthy volunteers (6 males and 4 females; age: 27–60 years [39.8 ± 3.1 (mean ± SD)]) were enrolled in the study and were anonymized randomly as A–G, O, Q, and R (Table 1). The inclusion criteria were as follows: healthy men and women over 20 years of age. The exclusion criteria were as follows: daily smoking; treatment with local or systemic antibiotics within 1 month prior to the study; and allergy to kiwifruit. The method and objective of this study were explained to the participants, who provided written informed consent before participating. The Osaka Dental University Medical Ethics Committee approved this study (approved on 3/31/2015; approval number 110864) and the investigations were conducted following the rules of the Declaration of Helsinki. The committee did not consider the study to be interventional in nature and therefore is not a clinical trial.\n\nTwo types of oral care tablets for tongue cleaning were tested in the current study. One type contained actinidin, a cysteine protease extracted from kiwifruit (“protease tablet”) and the other did not (“plain (placebo) tablet”). Both tablets were provided by Ezaki Glico Co., Ltd (Osaka, Japan). The protease tablets are identical to those marketed as BREO EX (Ezaki Glico Co.). Tablet composition was described previously12. To use the tablets, the participants placed one tablet on the dorsum of the tongue and waited until it dissolved naturally. One tablet takes approximately 5–7 min to completely dissolve.\n\nThe study design is illustrated in Figure 1. The tongue tablet experiment was a placebo-controlled double-blind crossover study. The 10 participants were randomly divided into 2 groups of 5 participants each, by using computer-generated random numbers. All participants performed an initial tongue cleaning (by brushing) at the beginning of the study. The participants were asked not to eat, drink, or perform oral cleaning before each sampling. After a washout period of 10 days during which the participants did not perform any tongue cleaning, they collected their saliva and tongue coating into separate containers in the morning immediately after waking up (sample D1). Then, the participants in each group took tablets, with or without the protease. The participants and the researchers who analyzed the data were not informed about the tablet types given to the participants. The participants were asked to use the tablet three times on the day of the experiment—in the morning (between 9–12 am), in the afternoon (1–4 pm), and in the evening (7–10 pm)—taking one tablet each time. The following morning, the participants collected their saliva and tongue coating separately immediately after waking up (sample D2). After a washout period of 10 days, the participants took the other type of tablet that they had not previously received, and collected the saliva and tongue samples as before. Control experiments (no tablet usage) were conducted with the same participants, after they conducted treatment using the tablets. The duration between the tablet treatments and the control experiment ranged from 10–60 days, depending on the participant. In these experiments, after an initial tongue cleaning and a 10-day washout period, all participants collected samples on two consecutive days (D1 and D2) without taking any tablet in between.\n\n(a) Ten participants were randomly divided into two groups for the tongue tablet trials. (b) Control (no tablet usage) treatment. The duration between the tablet treatments and the control experiment ranged from 10–60 days.\n\nThe saliva and tongue-coating samples were collected immediately after the participants woke up, in the morning of the day of tongue cleaning by tablet (D1) and the next morning (D2). The participants first collected 3 mL of saliva in a 25-mL sterile plastic tube. The tongue coating was collected by scrubbing the tongue with a swab and then soaking the tip of the swab in 0.6 mL of phosphate-buffered saline (PBS(-); Wako Pure Chemical Industries, Ltd., catalogue number 166-23555) to suspend the coating. Because this collection method involves scrubbing the tongue with a swab, the tongue coating was collected from the left half of the tongue for D1 and from the right half of the tongue for D2, to minimize the carryover effects of scrubbing. The collected samples were maintained at 4°C for up to 1 day and transported to the laboratory. The saliva samples (3 mL) were homogenized by repetitive pipetting. Then, 0.5-mL aliquots were transferred to sterile tubes. The saliva (0.5 mL) and tongue-coating (0.6 mL) samples were then centrifuged at 10,000 × g for 4 min. The supernatant was discarded and the pellet was stored at −20 °C until DNA extraction. All samples were frozen no later than on the day of D2 sampling.\n\nBacterial DNA was extracted from the pellets via chemical and mechanical lysis using a QIAamp UCP Pathogen Mini kit (QIAGEN, catalogue number 50214), as previously described17,18. Briefly, thawed pellets were immediately suspended in 0.5 mL of ATL buffer containing the optional DX reagent, transferred to a Pathogen Lysis Tube S, and then homogenized using a Mixer Mill MM 301 (Retsch) for 3 min at a vibrational frequency of 30 Hz. The manufacturer’s protocol was followed thereafter to complete the DNA purification. DNA was eluted in 50 μL of the AVE buffer (QIAGEN). DNA concentration was determined using a Quantus fluorometer (Promega) and a Qubit dsDNA BR Assay kit (Thermo Fisher Scientific, catalogue number Q32850). DNA was stored at –80 °C until use.\n\nBacterial 16S ribosomal DNA amplification and library construction were performed according to the 16S Metagenomic Sequencing Library Preparation guide supplied by Illumina (part No. 15044223_B), as previously described17. The V3–V4 region of the 16S ribosomal RNA gene was amplified by polymerase chain reaction (PCR) with a thermal cycler MJ-Mini (Bio-Rad Laboratories), using primers 341F (5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3’) and 806R (5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGACTACHVGGGTWTCTAAT-3’) (custom-synthesized by Invitrogen), and Premix Ex Taq Hot Start Version (Takara Bio, catalogue number RR030A). The thermal cycling conditions were initial denaturation at 98 °C for 10 s, followed by 25 cycles at 98 °C for 10 s, 55 °C for 30 s, and 72 °C for 1 min (the first PCR step). The underlined nucleotides served as primer sequence parameters to extract the V3–V4 region for feature classifier training (see next section). The amplicons were purified using AMPure XP beads (Beckman Coulter, catalogue number A63880). Sequencing adapters containing 8-bp indices were incorporated at the 3’- and 5’-ends of the purified amplicons during a second PCR step. The amplicons were again purified using the AMPure XP beads, and then quantified using a Quantus fluorometer (Promega) and a Qubit dsDNA HS Assay kit (Life Technologies, catalogue number Q32851). After pooling equimolar amounts of the amplicons, 5% of an equimolar amount of PhiX DNA (PhiX Control v3, Illumina, catalogue number FC-110-3001), was added. The obtained library was pair-end sequenced at 2 × 250 bp using a MiSeq Reagent Kit v2 (Illumina, catalogue number MS-102-2001) and the Illumina MiSeq platform. Sequencing was performed over seven independent runs at the Oral Microbiome Center (Takamatsu, Japan), followed by demultiplexing. Raw nucleotide sequences are available at DDBJ/EMBL-EBI/NCBI database under the accession number DRA010849.\n\nDemultiplexed paired-end sequences were processed using QIIME 2 (v.2020.2) and its associated plugins19 in a Docker container. Sequences obtained from independent Miseq runs were denoised separately using DADA2 (via q2-dada2)20 applying previously-optimized parameters14 (trim-left-f = 20; trim-left-r = 20; trunc-len-f and trunc-len-r were set between 241 and 248 depending on the sequence quality; other parameters followed the default settings, including chimera-method = “consensus”). The resulting exact amplicon sequence variants (ASVs) were merged (via q2-feature-table). For taxonomy assignment to each ASV, a naïve Bayes taxonomy classifier trained (via q2-feature-classifier)21 on the V3–V4 region of the 16S rRNA sequences in the expanded human oral microbiome database (eHOMD; v.15.2)22 was used. All ASVs were aligned using MAFFT23 and used to construct a phylogeny with FastTree 2 (via q2-phylogeny)24. Sample metadata format was validated using the cloud-based tool Keemei25.\n\nAlpha diversity was assessed by calculating the number of observed features (ASVs) and the Shannon index (via q2-diversity), after samples were subsampled without replacement (rarefied) to 40,000 sequences per sample. The non-parametric Kruskal–Wallis test was used to test for significant differences in alpha diversity between sample groups (P < 0.05).\n\nBeta diversity was computed based on the unweighted UniFrac distance26 (via q2-diversity) and visualized as three-dimensional principal coordinate analysis (PCoA) plots using EMPeror (via q2-emperor)27. Permutational analysis of variance (PERMANOVA)28 was used to test the significant difference in bacterial composition among samples (P < 0.05). Differential abundance of bacterial taxonomic groups was tested using the analysis of composition of microbiomes (ANCOM) (via q2-composition)29.\n\nClustered operational taxonomic unit (OTU) table was also used to calculate the beta diversity and differential abundance of specific taxonomic groups. Open-reference clustering was chosen for a high-quality taxonomic assignment to a curated database30. Here, the entire ASVs were clustered into OTUs by open-reference picking (via q2-vsearch)30 using the V3–V4 region of 16S rRNA sequences in the eHOMD v.15.2 as a reference, with 99% identity threshold. A phylogenetic tree was constructed as described above. The R package treeio (v.1.12.0)31 was used to change the OTU names within the phylogenetic trees, as per the requirement for the Rhea package (the software disallows OTU names starting with a number). Multidimensional scaling (MDS) based on generalized UniFrac distance32 was performed to examine the difference in microbial composition among samples, using Rhea pipeline (v.1.1.3)33. For differential abundance analysis, “Serial group comparisons” in Rhea was performed (abundance_cutoff = 0.2; prevalence_cutoff = 0.3; max_median_cutoff = 1) with significance cutoff of p < 0.05 in Kruskal-Wallis test.\n\nBox plots that show the alpha diversities were generated using R packages ggplot2 (v.3.3.2) and ggpubr (v.0.4.0), with data retrieved from QIIME 2 artifacts using qiime2R (v.0.99.31). Heatmaps and box plots that show the relative abundance of microbial taxonomies, and a Venn diagram that show core OTUs, were generated using ampvis2 (v.2.6.5)34. R (v.4.0.2) and RStudio (v.1.3.959) were used for all analyses. Software, plugins, and R packages used in this study is listed in Table 2.\n\nQIIME 2 plugins are not listed here because they are associated with specific version of QIIME 2.\n\n\nResults\n\nThe study design is illustrated in Figure 1. Ten healthy adults participated in the study, with three different treatments tested: two different types of oral tablets (with or without protease), and a negative control (no tablet). For the tablet treatments, the saliva and tongue coating were collected before (D1) and after (D2) the intervention. Overall, 116 samples were collected [10 participants, three treatments, two sites (the saliva and tongue), and two sampling time points (D1 and D2), with four samples excluded because of insufficient amount of extracted DNA]. The sample metadata are provided as underlying data (Table S1)35.\n\nDNA was extracted from each sample and the V3–V4 region of the 16S rRNA gene was PCR-amplified. The amplicons were paired-end sequenced using the Miseq platform. After quality control and error correction using DADA2, 11,260,102 reads corresponding to 5342 ASVs were obtained. Per-sample median was 89,923, with a maximum of 186,864 and a minimum of 46,422. Open-reference clustering, using the curated 16S rRNA sequences in the eHOMD v.15.2 database as the reference (at 99% identity threshold), grouped the sequences into 1210 OTUs. Either the full or clustered table was analyzed further, depending on the type of analysis performed, as described. The clustered OTU table is provided as underlying data (Table S2)36.\n\nWe first analyzed the microbiome of the saliva and tongue coating, to determine the baseline for the study. In total, 30 D1 samples (10 participants, three independent treatments) each of the saliva and tongue coating were analyzed. Alpha diversity of the tongue microbiome was significantly lower than that of the salivary microbiome, using both the number of observed ASVs (P = 8.9e-7, Kruskal–Wallis test) (Figure 2a) and Shannon index (P = 2.0e-7, Kruskal–Wallis test) as measures (Figure 2b). This was consistent with a previous report3. The difference in diversity is probably associated with the tongue acting as a specialized niche for specific microorganisms, and the saliva containing a mixture of microbiota from different sites in the oral cavity. Interestingly, the number of observed ASVs varied among the individuals, ranging from approximately 170 to 325 in the saliva (P = 0.027, Kruskal–Wallis test), and from approximately 125 to 225 in the tongue coating (P = 0.022) (Figure 2c), suggesting a difference in oral microbiome among individual.\n\nWe next assessed the differences in bacterial composition among samples (beta diversity) (Figure 3). A significant difference between the salivary and tongue microbiomes was detected both in PCoA based on unweighted UniFrac distances using the full ASV table (P = 0.001, PERMANOVA) (Figure 3a) and MDS based on generalized UniFrac distances32 using the clustered OTU table (P = 0.003, PERMANOVA) (Figure 3c).\n\nData from the full amplicon sequence variants (ASV) table were used to calculate the alpha-diversity indexes. (a, b) Thirty samples each from the salivary or tongue microbiome were compared (three D1 samples for each of 10 participants). The number of observed ASVs (a) or Shannon index (b) was used as the alpha-diversity measure. Each point indicates a sample. Colors of the points indicate different participants. (c) Box plots of the number of observed ASVs in the salivary and tongue microbiomes (n = 3 each) for each participant.\n\n(a, b) Three-dimensional principle co-ordinate analysis (PCoA) plots were generated with EMPeror using the full amplicon sequence variants (ASV) table. While only samples corresponding to D1 are displayed (n = 58), the PCoA analysis included all (116) samples. (c, d) multidimensional screening (MDS) plot of microbial profiles calculated based on generalized UniFrac distances using the clustered operational taxonomic unit (OTU) table. Each point indicates a sample. The points are colored according to the sampling site (a, c) or the participant from whom the sample was obtained (b, d).\n\nThe data also revealed a significant difference between individual microbiomes (Figure 3b and 3d; P = 0.001, PERMANOVA using the clustered OTU table). As indicated by the plots in Figure 3, the similarity of the salivary and tongue microbiomes within an individual was greater than the similarity of the salivary or tongue microbiomes between individuals. This suggests there is stability in an individual’s oral microbiome, at least within the relatively short time period of the study (several weeks). This observation is consistent with earlier studies that highlight the stability of an individual’s oral microbiome37.\n\nThe abundances of bacterial taxonomic groups at the genus or species levels in the salivary and tongue microbiomes, determined by the analysis of D1 samples from the three treatments and based on the clustered OTU table are summarized in Figure 4a and 4b. The eight most abundant genera were common to the salivary and tongue microbiomes, accounting for nearly 80% of both microbiomes (78.2% in the saliva and 80.9% in the tongue). These genera were Prevotella (18.4% and 23.5%, respectively), Veillonella (9.0% and 12.6%, respectively), Neisseria (11.6% and 9.9% respectively), Haemophilus (10.9% and 8.6%, respectively), Streptococcus (9.4% and 6.1%, respectively), Alloprevotella (8.2% and 5.9%, respectively), Porphyromonas (6.1% and 6.7%, respectively), and Fusobacterium (4.6% and 7.6%, respectively) (Figure 4a). The abundance of bacterial taxa at species level is shown in Figure 4b. Prevotella melaninogenica HMT-469 was most abundant in both microbiomes (8.1% in the saliva and 11.8% in the tongue), followed by Streptococcus oralis subsp. dentisani HMT-398 (7.2%) and Haemophilus parainfluenzae HMT-718 (7.0%) in the saliva, and by Fusobacterium periodonticum HMT-201 (7.4%) and H. parainfluenzae HMT-718 (7.4%) in the tongue (Figure 4b).\n\nFive OTUs were differentially abundant in the salivary and tongue microbiomes (P < 0.05, Kruskal–Wallis test) (Figure 4c). Among them, S. oralis subsp. dentisani HMT-398 (7.2% in the saliva and 2.7% in the tongue) and Neisseria mucosa HMT-682 (1.8% and 0.2%, respectively) were more abundant in the saliva, whereas F. periodonticum HMT-201 (3.5% and 7.5%, respectively), P. melaninogenica HMT-469 (8.1% and 11.8%, respectively), and Prevotella histicola HMT-298 (1% and 2.1%, respectively) were more abundant in the tongue (Figure 4b and 4c).\n\nThe clustered operational taxonomic unit (OTU) table was used for calculations. (a, b) Heatmaps of the abundance of bacterial taxa at the genus (a) or species (b) levels. The % read abundances are indicated, together with a color gradient. (c) Box plots summarizing the abundance of OTUs that were differentially abundant (P < .05, Kruskal–Wallis test with Benjamini–Hochberg adjustment) in the saliva and tongue samples at the species level are shown. Each dot indicates a sample. Taxon IDs in eHOMD are indicated in the parentheses.\n\nTo identify the core members of the oral microbiome, we focused on OTUs that were present in ≥95% of the saliva or tongue D1 samples. Seven OTUs were present in ≥95% of both, the saliva and tongue samples. These were F. periodonticum HMT-201, Saccharibacteria (TM7) [G-1] sp. HMT-352, S. oralis subsp. dentisani HMT-398, P. melaninogenica HMT-469, Granulicatella adiacens HMT-534, Campylobacter concisus HMT-575, and H. parainfluenzae HMT-718 (Figure 5).\n\nOTUs present in ≥95% of samples (D1 samples, n = 58) in the indicated subset of oral sites are shown together with the average total abundance of the OTUs in the group. For example, Veillonella parvula was found in ≥95% of saliva samples and <95% of tongue samples. Taxon IDs in eHOMD are indicated in the parentheses.\n\nFurther, we identified site-specific core OTUs, detected in ≥95% samples from one site but not from the other. The saliva-specific core OTUs were Veillonella parvula HMT-161, Porphyromonas pasteri HMT-279, Prevotella nanceiensis HMT-299, Gemella haemolysans HMT-626, and Alloprevotella sp. HMT-914. The tongue-specific core OTUs were Catonella morbi HMT-165, Oribacterium sinus HMT-457, Peptostreptococcaceae [XI][G-1] sulci HMT-467, Solobacterium moorei HMT-678, and Rothia mucilaginosa HMT-681 (Figure 5).\n\nUsing the above data as the base line, we finally assessed the effect of taking oral tablets (with or without protease) on the salivary and tongue microbiomes. Alpha diversity in D1 and D2 samples was not significantly different between any treatments (Figure 6a). In the control (no tablet) treatment, whereas the observed number of ASVs seemed to slightly increase in the saliva and to slightly decrease in the tongue, they were not statistically significant (Figure 6a). This indicates that some fluctuation of the oral microbiome may occur naturally. Further, MDS analysis indicated that the beta diversity between D1 and D2 samples was not significantly different in any treatment, for either the salivary or tongue microbiome (P > 0.7, Kruskal–Wallis test) (Figure 6B).\n\n(a) Box plots of the number of observed amplicon sequence variants (ASV) in the saliva and tongue D1 and D2 samples in the no tablet (left), protease tablet (middle), and plain tablet (right) treatments. Each point indicates a sample. (b) multidimensional screening (MDS) analysis of beta diversity in D1 and D2 samples in the saliva (top panels) and tongue (bottom panels) in no tablet (left), protease tablet (middle), and plain tablet (right) treatments.\n\nWe next examined whether any bacterial species were specifically impacted by oral tablet usage. Both ANCOM using the full ASV table or Kruskal–Wallis test using the clustered OTU table revealed that no OTU was differentially abundant before (D1) or after (D2) tablet use, in any of the treatments (no tablet, protease tablet, and plain tablet). The OTU abundance in each treatment group is summarized in Figure 7a–c. Although according to a recent study oral tablet use decreases the abundance of Fusobacterium nucleatum on the tongue of healthy young adults16, we did not detect any significant decrease of OTUs that correspond to F. nucleatum. Further, in the current study, whereas 7.6% of all OTUs from the tongue microbiome were assigned to the genus Fusobacterium (Figure 4a), the majority of them were classified as F. periodonticum (7.5% of total) and only <0.1% of all OTUs was assigned to F. nucleatum at species level.\n\nThe abundances of top 12 operational taxonomic units (OTUs) based on the clustered OTU table for the no tablet (a), protease tablet (b), and plain tablet (c) treatments are shown. The data are colored depending on the group (saliva or tongue, and D1 or D2). No bacterial species showed differential abundance before (D1) or after (D2). Taxon IDs in eHOMD are indicated in the parentheses.\n\n\nDiscussion\n\nThe oral microbiota has been associated with specific diseases in susceptible populations. In the current study, we examined the effect of oral care tablet use, with or without actinidin, on the salivary and tongue microbiomes. We showed that whereas there are some differences between the tongue and salivary microbiomes, the microbiomes were not affected by the oral tablet use, regardless of the tablet type. This does not preclude the possibility that a persistent oral tablet use would alter the oral microbiome. Controlled alteration of the oral microbiome has potential for disease prevention.\n\nWe here identify the core OTUs that are common between saliva and tongue. Among these OTUs, S. oralis and Campylobacter sp. have been determined to be the core OTUs common in the saliva and tongue previously3. F. periodonticum and Granulicatella adiacens have been found in tongue microbiome of adult, P. melaninogenica and H. parainfluenzae have been found in infant and adult tongue in common38. TM7 species have been identified in oral microbiomes including tongue39,40 and supragingival plaque41. It should be noted that although the high prevalence of Saccharibacteria (TM7) [G-1] sp. HMT-352 (≥95% in both saliva and tongue) as shown in our present study has not been reported previously, it could also be a result of clustering similar sequences into a single OTU. Although there are some differences in the classification of the core or predominant oral OTUs between the current and other studies1,3, the majority of the species were identified as the core oral OTUs across the studies. Since low-abundance rather than highly abundant OTUs may contribute more to the difference in oral bacterial communities42,43, detailed analysis of low-abundance OTUs would be important in future research. O. sinus and S. moorei were previously classified as core OTUs common to the salivary and tongue microbiomes3, but in our present study were identified as tongue-specific. This seems reasonable considering that S. moorei plays an important role in halitosis44–46. The effective separation of saliva- and tongue-specific OTUs suggest the usefulness of our study design in analyzing the salivary and tongue microbiomes simultaneously.\n\nOur present study shows a variety among individuals in the number of observed ASVs in the salivary and tongue microbiomes. On the contrary, a significant interpersonal diversity in the supragingival plaque and salivary microbiomes, but not in the tongue plaque microbiomes was previously reported, using Faith’s phylogenetic diversity as a measure3. Since the same V3–V4 region was targeted for amplicon sequencing in both studies, the discrepancy concerning the interpersonal differences in tongue microbiome might be associated with the differences in the alpha-diversity measure used, and/or in the participants’ age [25.3 ± 3.1 years in Hall et al. study3 and 39.8 ± 10.9 years (mean ± SD) in the current study (see Methods)].\n\nThe tongue microbe is associated with various diseases, including halitosis47,48 and aspiration pneumonia4; alterations in salivary microbiome has also linked to increasing numbers of oral and non-oral diseases49. With the advancement of microbiome studies, methods to predict host traits that predispose to various diseases or conditions based on microbiome analysis have been developed50,51. Lu et al. have shown that tongue coating microbiome data can be used to distinguish individuals with pancreatic head carcinoma (PHC, one of pancreatic adenocarcinoma which occurs in the head of the pancreas) from healthy subjects39. Although the evaluation of an individual’s disease status based on the tongue microbiota data is possible, the collection methods of the tongue coating samples may not be reliable when performed by a non-specialist, especially because of the anterior to posterior gradient of the bacterial communities in the tongue surface43. We here showed that the microbiomes of the saliva and tongue of an individual tend to be more similar to one another than to the salivary or tongue microbiomes from other individuals. Considering this fact together with the stability of oral microbiome over a prolonged period of time37, salivary collection could perhaps be used in the future as a standard method to predict diseases associated with the tongue-coating microbiota, as well as those linked to that of the saliva.\n\nOral care tablets have been shown previously to reduce tongue coating load and VSCs12,16. We here analyzed the effect of oral care tablets on the salivary and tongue microbiome. To avoid individual varieties in the amount of tongue coating or saliva flow affecting the analysis, we recruited only healthy adults to participate in this study. We did not detect any significant differences in the alpha diversity, beta diversity, or abundance of specific OTUs at species level after oral tablet use. There are several possible explanations for these observations. First, the participants of the current study were healthy adults with no apparent tongue coating accumulation. Although accumulated tongue coating can be reduced with oral tablets12, the amount of tongue coating analyzed herein may have been insufficient for detecting the differences in the microbiota. Second, the tablet intervention period in the current study was only 1 day and the samples were collected 1 day after the tablet use. In contrast, twice daily tongue scraping for three days, together with sampling within 15 mins after intervention, have shown to reduce the gram-negative anaerobes on the tongue9. Although we chose to collect samples 1 day after the intervention, the 1-day period could have been long enough for the resilience of the oral microbiota to revert any shift in the oral microbiomes invoked by the tablet use. Considering these factors, analyzing oral care tablet intervention in individuals with a higher tongue coating index and/or over a longer period of time together with immediate sampling after intervention may provide more information on whether and how oral care tablets alter the oral microbiota, contributing to the maintenance of oral health.\n\nOral care tablets containing actinidin reduces tongue coating, and actinidin prevents biofilm formation by degrading cell-surface proteins in vitro12. We here attempted to elucidate the effect of the protease, supplied in oral tables, on the oral microbiome. Unfortunately, we were unable to assess the effect of the protease, because oral tablet treatments failed to alter the oral microbiome or specific bacterial taxa, regardless of the presence or absence of actinidin. As above, including participants with a higher tongue coating and a longer intervention period with immediate sampling may have allowed detection of the effect of actinidin in the tablets. Alternatively, an in vitro culturing system could be used to analyze the effect of actinidin on the oral microbiome, with the effects of the compound tested in a controlled manner. For example, nitric oxide17 or statins52 have been shown to alter the abundance of specific bacterial species. Using such system would allow the analysis of the effect of actinidin on oral microbiota separately from the effect of mechanical removing of the tongue coating.\n\nVarious lines of evidence suggest a link between oral microbiota and health or disease1,2,53. The current and other3 studies have highlighted interpersonal differences in the oral microbiota. Several types of tongue microbiota have been shown to exist in individuals with different susceptibility to pneumonia4. Hence, personalized treatment based on an individual’s oral microbiota is required, as has been already pointed out in the context of periodontal disease54. Analysis of how different types of oral microbiota are affected by certain interventions (e.g., oral care tablet or antibiotic treatment) would enable a more precise control over the oral microbiome in the future. In vitro culturing systems mentioned above are powerful tools for elucidating responses of bacterial communities taken from different individual to various interventions, and the contributing factors.\n\nIn conclusion, we have shown that while the salivary and tongue microbiomes differ significantly in terms of bacterial composition, they show inter- rather than intra-individual diversity. We have also identified bacterial species that are common to the salivary and tongue microbiome, as well as those that are specific to either of these. In addition, we showed that oral care tablets may not alter the bacterial composition of the saliva or the tongue, at least over short periods of time in healthy individuals. Considering the link between oral microbiota and health or disease, analyzing the differences in how individual oral microbiota responds to external factors will pave the way to more effective therapeutic and diagnostic approaches and, ultimately, contribute to the development of personalized dental medicine.\n\n\nData availability\n\nRaw nucleotide sequences are available at DDBJ/EMBL-EBI/NCBI database under the accession number DRA010849.\n\nFigshare: Table_S1_sample-metadata.tsv for \"Inter-site and interpersonal diversity of salivary and tongue microbiomes, and the effect of oral care tablets\". https://doi.org/10.6084/m9.figshare.13289618.v135\n\nTable_S1_sample-metadata.tsv: Columns indicate sample ID, participant, sampling date (D1 or D2), treatment, Miseq run number, and sampling body site (saliva or tongue) of each sample. The “treatment” column indicates, no tablet (E2), protease tablet (E3), or plain tablet (E4) treatments.\n\nFigshare: Table_S2_clustered-OTU-table.tsv for \"Inter-site and interpersonal diversity of salivary and tongue microbiomes, and the effect of oral care tablets\". https://doi.org/10.6084/m9.figshare.13291535.v136\n\nTable_S2_clustered-OTU-table. After open-reference clustering, OTU table was constructed from the BIOM file using QIIME 2. Number of reads for each OTU in each sample are indicated, together with the bacterial taxonomy assigned to each OTU. OTU IDs are identical to matching HOMD Refseq IDs, except for those which did not match the database.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgments\n\nWe would like to thank Hisako Sakamoto for preparation of DNA libraries, Makoto Taniguchi for Miseq sequencing, and Editage (www.editage.com) for English language editing.\n\n\nReferences\n\nYamashita Y, Takeshita T: The oral microbiome and human health. J Oral Sci. 2017; 59(2): 201–206. PubMed Abstract \| Publisher Full Text\n\nWillis JR, Gabaldon T: The Human Oral Microbiome in Health and Disease: From Sequences to Ecosystems. Microorganisms. 2020; 8(2): 308. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHall MW, Singh N, Ng KF, et al.: Inter-personal diversity and temporal dynamics of dental, tongue, and salivary microbiota in the healthy oral cavity. NPJ Biofilms Microbiomes. 2017; 3: 2. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "76292", "date": "22 Dec 2020", "name": "Takuichi Sato", "expertise": [ "Reviewer Expertise Oral Microbiology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSuggestions:\nResults Figure 4(b): The authors need to check the figure. There are THREE \"Porphyromonas pasteri (279)\" in the figure.\nDiscussion Figure 5 should be cited in the second paragraph of the discussion.\n\nTypographical errors; Results P. 6: \"Neisseria (11.6% and 9.9% respectively)\" Insert a comma between \"9.9%\" and \"respectively\".\nP. 7: \"F. periodonticum HMT-201 (3.5% and 7.5%, respectively.\" \"7.5%\" should read \"7.4%\".\nFigure 4(a): The authors need to check the figure. Absconditabacteria?\nReferences: The authors need to check the style of the references #2, 4, 7, 13, 17, 22, 26, 31, 37, 38, 41, 44, 50, 51 and 54. (Do not use capital letters in the title of each reference.)\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6529", "date": "09 Apr 2021", "name": "Hugo Maruyama", "role": "Author Response", "response": "Thank you very much for your review and comments on our manuscript. Below is a point-by-point response to your comments. Suggestions: Results Figure 4(b): The authors need to check the figure. There are THREE \"Porphyromonas pasteri (279)\" in the figure. Thank you for pointing this out. This was due to the multiple slightly different reference 16S rRNAs sequences present in the HOMD database used for open-reference clustering of ASVs into OTUs (http://www.homd.org/index.php?name=HOMD&view=dynamic&oraltaxonid=279). Because the original figure was created based on counts per OTU, the table contained three \"Porphyromonas pasteri (279)\". In the revised figure, we aggregated the counts for OTUs with a common Taxon ID (in this case, HMT-279). An explanation was added to the figure legend: \"In (b), count for clustered OTUs with common Taxon ID were aggregated.\" Discussion Figure 5 should be cited in the second paragraph of the discussion. Figure 5 is now cited as recommended. Typographical errors; Results P. 6: \"Neisseria (11.6% and 9.9% respectively)\" Insert a comma between \"9.9%\" and \"respectively\". P. 7: \"F. periodonticum HMT-201 (3.5% and 7.5%, respectively.\" \"7.5%\" should read \"7.4%\". All typographical errors that were pointed out have been corrected. Figure 4(a): The authors need to check the figure. Absconditabacteria? The problem of the left edge of the figure being cut off has been fixed. References: The authors need to check the style of the references #2, 4, 7, 13, 17, 22, 26, 31, 37, 38, 41, 44, 50, 51 and 54. (Do not use capital letters in the title of each reference.) The style of the references has been unified as pointed out." } ] }, { "id": "79337", "date": "01 Mar 2021", "name": "Rohit Kunnath Menon", "expertise": [ "Reviewer Expertise oral microbiome" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nComment 1 Abstract: The results section should provide values for the diversity and the names of at least the most important core OTUs.\nComment 2 The authors may include a significant limitation of the study in the discussion section. Which is the sample size of ten individuals. The conclusions regarding the inter-participant differences rather than intra-individual variation is also previously well-established by studies with larger sample size. Hence it is ambitious to claim that this result is well-established with the small sample size.\nComment 3 Another significant limitation is the the lack of clarity in how the individuals were deemed healthy orally as well as systemically. . The absence of clinical data on the caries and periodontal health status of each participant needs to be explained. Previous research clearly shows the impact of oral diseases in determining the microbiome of the oral cavity especially saliva.\nComment 4 It is not surprising that one day treatment with the tablet did not significantly impact the temporal variation. Previous research has shown that even antibiotic treatment does not significantly impact salivary microbiome. The impact of the external agents on the microbiome should be discussed with inclusion of more of such previous investigations. The exclusion criteria of one month for antibiotic treatment should also be discussed with respect to previous available literature:\nR K Menon, A Gomez, B W Brandt, Y Y Leung, D Gopinath, R M Watt, W Crielaard, K E Nelson, M G Botelho. Long-term impact of oral surgery with or without amoxicillin on the oral microbiome-A prospective cohort study. Sci Rep. 2019 Dec 10;9(1):187611\n\nZaura, E. et al. Same Exposure but Two Radically Different Responses to Antibiotics: Resilience of the Salivary Microbiome versus Long-Term Microbial Shifts in Feces. mBio 6, e01693–01615 (2015)2\nComment 5 Please check the English grammar. For example: \"We here analyzed the effect of oral care tablets on the salivary and tongue microbiome.\"\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6528", "date": "09 Apr 2021", "name": "Hugo Maruyama", "role": "Author Response", "response": "Thank you very much for your review and comments on our manuscript. Below is a point-by-point response to your comments. Comment 1 Abstract: The results section should provide values for the diversity and the names of at least the most important core OTUs. We have included the values of alpha diversity and the names of the core OTUs in the abstract. Comment 2 The authors may include a significant limitation of the study in the discussion section. Which is the sample size of ten individuals. The conclusions regarding the inter-participant differences rather than intra-individual variation is also previously well-established by studies with larger sample size. Hence it is ambitious to claim that this result is well-established with the small sample size. The following statement has been added to the last paragraph of the Discussion: \", although it should be noted that the study has a limitation in the sample size of ten individuals.\" Comment 3 Another significant limitation is the lack of clarity in how the individuals were deemed healthy orally as well as systemically. The absence of clinical data on the caries and periodontal health status of each participant needs to be explained. Previous research clearly shows the impact of oral diseases in determining the microbiome of the oral cavity especially saliva. The following description has been added to the \"Participants\" section of the Methods: \"According to the medical questionnaire, (1) none of the participants were undergoing or planning treatment for dental caries or periodontal disease, (2) there were no participants who were suffering from diabetes, chronic kidney disease, lung diseases, malignant tumors, etc., or who were visiting hospitals or taking medication, and (3) none of the participants experienced frequent thirst.\" Comment 4 It is not surprising that one day treatment with the tablet did not significantly impact the temporal variation. Previous research has shown that even antibiotic treatment does not significantly impact salivary microbiome. The impact of the external agents on the microbiome should be discussed with inclusion of more of such previous investigations. The exclusion criteria of one month for antibiotic treatment should also be discussed with respect to previous available literature: R K Menon, A Gomez, B W Brandt, Y Y Leung, D Gopinath, R M Watt, W Crielaard, K E Nelson, M G Botelho. Long-term impact of oral surgery with or without amoxicillin on the oral microbiome-A prospective cohort study. Sci Rep. 2019 Dec 10;9(1):187611 Zaura, E. et al. Same Exposure but Two Radically Different Responses to Antibiotics: Resilience of the Salivary Microbiome versus Long-Term Microbial Shifts in Feces. mBio 6, e01693–01615 (2015)2 The following description has been added to the \"Participants\" section of the Methods: \"The exclusion criteria of one month for antibiotic treatment was set based on previous reports on the robustness and resilience of salivary microbiome [Zaura, E. et al. 2019;Menon RK et al. 2015]. For example, change in microbiome caused by exposure to clindamycin lasted up to 1 month in saliva [Zaura, E. et al.].\" In addition, the following statement has been added to the sixth paragraph of the Discussion: \"The impact of external agents on microbiome depends on the location of microbiome. For example, salivary microbiome is highly resilient against external agents including antimicrobials, compared to feces microbiome that is more easily affected [Zaura et al. mBio 6, e01693–01615 (2015)2].\" Comment 5 Please check the English grammar. For example: \"We here analyzed the effect of oral care tablets on the salivary and tongue microbiome.\" The text has been changed to \"Here, we analyzed the effect of oral care tablets on the salivary and tongue microbiomes.\" The entire manuscript has been checked by a native speaker." } ] }, { "id": "79136", "date": "01 Mar 2021", "name": "Priya Nimish Deo", "expertise": [ "Reviewer Expertise Oral microbiome and oral cancer" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIt is a well designed study. Effect of one day use of oral care tablets on oral microbiome was studied. Monitoring of the salivary and the tongue microbiomes is conducted simultaneously during an interrvention. Their results showed that the alpha diversity was higher in the saliva than on the tongue without intervention. The tablets did not affect the diversity not the abundance of specific species. Studies need to be carried out for longer duration to study the shifts in the composition of the microbiome upon intervention.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6527", "date": "09 Apr 2021", "name": "Hugo Maruyama", "role": "Author Response", "response": "Thank you very much for your review and comment on our manuscript. Studies need to be carried out for longer duration to study the shifts in the composition of the microbiome upon intervention. We agree that longer term study is needed to determine the impact of oral care tablets on the oral microbiome. This point is discussed in the \"Discussion\" section." } ] } ]	1	https://f1000research.com/articles/9-1477
https://f1000research.com/articles/9-1276/v1	27 Oct 20	{ "type": "Software Tool Article", "title": "SAPHIR: a Shiny application to analyze tissue section images", "authors": [ "Elodie Germani", "Hugues Lelouard", "Mathieu Fallet", "Hugues Lelouard", "Mathieu Fallet" ], "abstract": "Study of cell populations in tissues using immunofluorescence is a powerful method for both basic and medical research. Image acquisitions performed by confocal microscopy notably allow excellent lateral resolution and more than 10 parameter measurement when using spectral or multiplex imaging. Analysis of such complex images can be very challenging and easily lead to bias and misinterpretation. Here, we have developed the Shiny Analytical Plot of Histological Image Results (SAPHIR), an R shiny application for histo-cytometry using scatterplot representation of data extracted by segmentation. It offers many features, such as filtering of spurious data points, selection of cell subsets on scatterplot, visualization of scatterplot selections back into the image, statistics of selected data and data annotation. Our application allows to quickly characterize labeled cells, from their phenotype to their number and location in the tissue, as well as their interaction with other cells. SAPHIR is available from: https://github.com/elodiegermani/SAPHIR", "keywords": [ "Tissue cellular quantification", "spatial cellular profiling", "cell-cell interactions", "scatterplot", "histo-cytometry", "image cytometry" ], "content": "Introduction\n\nThe identification, localization and quantification of cell subsets in tissue is a difficult but essential task for biologists to understand spatial cellular organization in different settings (e.g. homeostasis vs inflammatory diseases or cancer). Advances in optical microscopy allow image acquisitions with more than 10 channel measurements using spectral fluorescence imaging or multiplex imaging combined with z-axis optical slices of tissue sections ranging from 10 µm to more than 200 µm when clearing methods are used1–4. Analysis of such complex images is very challenging due to the size and complexity of data. It requires image segmentation in 3D that can be further improved using deep learning-based segmentation5–7. Then, like flow cytometry, complex image analysis can benefit from scatterplot representations that allow to gate cells of interest2,8. Surprisingly, in existing software, this scatterplot representation is rarely interactive with the image itself, although this would allow to locate selection results back into the image but also to filter or correct results and fine-tune the gates defining cell populations to obtain in return a better visualization of them into the image.\n\nTo this end, we developed the Shiny Analytical Plot of Histological Image Results (SAPHIR), an R/Shiny application for the quantitative analysis of tissue section images. Since image segmentation is a complex task in continuous development and highly dependent on image quality and information, the integration of a single type of segmentation method in an image analysis application is not necessarily recommended. Therefore, we decided to separate segmentation from SAPHIR, but we provided two Fiji macro examples with associated images, which can be used to perform this task before running SAPHIR. SAPHIR offers many features such as interactive scatterplots with the image and data filtering and correction as described below.\n\n\nMethods\n\nThe segmentation required to use the application SAPHIR has been carried out under Fiji9, with custom-made macros, which are available on GitHub in the Demonstration Files. Cell nucleus detection was used for segmentation and fluorescence intensity signal of each channel within a circular ring around nuclei was determined to define cell of interest (COI) phenotype. In addition, regions of interest were defined using DBSCAN (optional)10. The segmentation process of the image to be analyzed should create a csv result file containing the channel intensity values (ranging from 0 to 255) and, if required, the positioning (x, y, z) linked to each COI. Another file, termed ROI.zip, should contain the image contour of each COI. Importantly, many other morphological measurements like area, roundness, or solidity as well as other information (COI belonging to a region of interest, COI interaction with other cells, COI centroid position) can be added to the segmentation result file. Finally, the minimal requirements to run the application are the image in TIFF with multiple channels and optionally slices, the segmentation result file with COI identity in csv and the roi.zip file containing contours of each COI. A legend file with all channel information can also be provided (optional).\n\nSAPHIR is built in the R programming language, version 4.0.2, and uses many packages available on CRAN (e.g. shinydashboard, ijtiff, magick, ggplot2, plotly) and one package (EBImage) available on Bioconductor. It has been tested on macOS and Windows 10. A user flowchart from image acquisition to obtention of quantitative data of COI with SAPHIR is shown in Figure 1.\n\nThe first step of SAPHIR is either to run a segmentation program to obtain appropriate files or to load the three required files (image.tif, roi.zip, results.csv) and if needed the legend (csv file) in the “select your results” menu of the application. Here, result, region of interest (ROI) and legend files were obtained with an in house-made segmentation macro developed under Fiji.\n\nThe first tab of the “Plot to Image” menu is shown in Figure 2 and allows the users to filter their data depending on one or two parameters displayed on histogram or scatterplot, respectively. This can be used to remove cells that have been badly segmented. Indeed, such cells display an aberrant area or volume (doublets or aggregates in red in Figure 2). Here, only cells within the Gaussian curve were retained (in blue in Figure 2). This can also be used to work only on given subpopulations filtered based on their location, interaction with other cells or other criteria (see use case section below). Then, filtered cells can be separated into subsets thanks to a two-parameter (most often channel intensities) scatterplot (Figure 3A). In addition, users can add a third parameter that is displayed on the scatterplot through a change of symbol shape for each COI beyond a threshold defined by the users for a given parameter value (Figure 3A).\n\nThe data-filtering tab allows the selection of cells to be analyzed based on one or two parameters. Based on the cell area parameter, only cells that displayed a conventional cell area (in blue) were selected. Badly segmented cells (large area in red) were discarded.\n\n(A) Scatterplot of two channel intensity parameters used to gate COI. User can add a third parameter that is displayed on the scatterplot through a change of symbol shape for COI beyond a threshold defined by the user. (B) Visualization of selected cells in the image. Some of the available options are shown (displayed channel(s), channel overlay, cell identity number, brightness).\n\nGating can be made easily with quadrants, rectangles and lassos in single or multiple selection mode. Interactivity between scatterplot and image is optional to avoid slow interaction when high-resolution images are used. In the latter case, we recommend users to perform their gating before allowing their selection to be shown in the image by ticking the appropriate box. Users can easily change the slice and the displayed channels with buttons to monitor selected COI spatial distribution and fluorescence intensity in the image (Figure 3B). Moreover, several options are available such as cell identity display or channel overlay (Figure 3B). Finally, statistics of the gated COI are downloadable, and gates are saveable for further analyses.\n\nThe SAPHIR menu “image to plot” allows to select a region in the image and to display cells of this region on a two-parameter scatterplot (Figure 4).\n\nSelection of one region in the image (left) and visualization of COI of this region in a corresponding scatterplot where two parameters are displayed (right).\n\nFinally, the menu “Annotation” allows, based on the previous analyses, correction of the data from the result csv file but also from the scatterplot-gated cells when saved in the “plot to image” menu. For each selected cell, a cropped image of this cell is displayed, and users can change its parameters if necessary (Figure 5).\n\nThe results to annotate can be selected based on the Plot to Image scatterplot gated cells that have been previously saved (left panel). Each cell of the selection can be visualized in the image (right panel) with the possibility to change image size (magnification), the displayed channel(s), the slice (z optical section) and the brightness. Based on the different analysis, results for the selected COI can be modified and final results saved and exported.\n\n\nUse case\n\nTo show the usefulness of our application, we used it on a project that aimed to characterize the interaction between phagocytes and proliferative immune effector cells in murine Peyer’s patches (PP), i.e. B and T cells. PP are immune inductive sites distributed along the small intestine in charge of sampling noxious antigens and mounting an immune response against them. In PP, antigens are taken up by phagocytes that, upon stimulation, migrate in the T cell zone and its periphery to interact with and prime naïve T cells. The periphery of the PP T cell zone is indeed an area of intense proliferation of immune effector cells after stimulation, suggesting that this region is a privileged site for their activation11. We therefore decided to examine the evolution of proliferative cell number, to determine their identity (B or T cells) and to analyze their interaction with migratory phagocytes during the course of the stimulation.\n\nWe used SAPHIR application and two ImageJ macros, which are available on GitHub in the Demonstration Files. The first macro allows the counting of proliferative cells through segmentation of Ki-67+ nuclei, provides their identity through analysis of T and B cell staining of Ki-67+ cell membrane, and analyses their interaction with phagocytes. The second macro identify the area of high density in proliferative cells as ROI, thanks to the DBSCAN algorithm and determine whether previously analysed cells belong to this ROI.\n\nThen, SAPHIR application allowed us to integrate these data and provided scatter plots and statistical analyses. Thus, the filtering tab was used to select only proliferative cells belonging to the ROI (Figure 6, upper left). Then, ROI-proliferative cells were split into B and T cells using the scatter plot (Figure 6, lower left). Finally, we used the “symbol shape change parameter” option to highlight ROI-proliferative cells that interacted with phagocytes. This clearly showed that in the ROI, there were more proliferative T cells than B cells that interacted with phagocytes. Exportable statistical tables confirmed this observation. Selecting these T cells, we could localize these cells interacting with phagocytes directly into the image (Figure 6, right).\n\nUpper left: Cells belonging to the proliferative area of the PP T cell zone were selected using the filtering tab. Lower left: ROI proliferative cells were split into T and B cells on the scatterplot and phagocyte-interacting cells highlighted thanks to the symbol shape change parameter (circle). Right: T cells selected in the scatter plot in the lower left (orange) are visualized in the image (magenta contour).\n\n\nConclusion\n\nThe SAPHIR application provides a simple and user-friendly interface to obtain quantitative data from tissue images as well as COI positioning in the tissue. It is based on the interactivity between quantitative data and image to simplify the analysis and limit bias. Further developments of SAPHIR will include use of pyramidal images to minimize loading and analysis time and unsupervised clustering methods for scatterplot generation12.\n\n\nData and availability\n\nSAPHIR is provided with segmentation data from two demo pictures (https://github.com/elodiegermani/SAPHIR/tree/master/Demonstration%20files): one with only two intensity channels to test the application in a very easy and quickly way, and a more complex one with 6 intensity channels, 12 slices and two additional parameters (belonging to a ROI and cell-cell interaction) for advanced testing of the application.\n\nThese data are parts of several projects conducted in the Center of Immunology of Marseille-Luminy (France). Acquisition of the images was made with spectral confocal microscopy and analyzed with Fiji with the provided macro.\n\n\nSoftware availability\n\nSource code available from: www.github.com/elodiegermani/SAPHIR\n\nArchived source code as at time of publication: http://doi.org/10.5281/zenodo.408889913\n\nLicense: GNU General Public License v3.0", "appendix": "Acknowledgments\n\nWe thank Guillaume Gay (CENTURI Multi-engineering platform) for helpful discussion; Mauro Gaya and Claude Grégoire for the use of an unpublished image.\n\nThis publication was supported by COST Action NEUBIAS (CA15124), funded by COST (European Cooperation in Science and Technology.\n\n\nReferences\n\nDodt HU, Leischner U, Schierloh A, et al.: Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain. Nat Methods. 2007; 4(4): 331–336. PubMed Abstract \| Publisher Full Text\n\nGerner MY, Kastenmuller W, Ifrim I, et al.: Histo-cytometry: a method for highly multiplex quantitative tissue imaging analysis applied to dendritic cell subset microanatomy in lymph nodes. Immunity. 2012; 37(2): 364–376. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGoltsev Y, Samusik N, Kennedy-Darling J, et al.: Deep Profiling of Mouse Splenic Architecture with CODEX Multiplexed Imaging. Cell. 2018; 174(4): 968–981.e915. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLelouard H, Mailfert S, Fallet M: A ten-color spectral imaging strategy to reveal localization of gut immune cell subsets. Zeiss Application note. 2018. Reference Source\n\nLegland D, Arganda-Carreras I, Andrey P: MorphoLibJ: integrated library and plugins for mathematical morphology with ImageJ. Bioinformatics. 2016; 32(22): 3532–3534. PubMed Abstract \| Publisher Full Text\n\nWeigert M, Schmidt U, Haase R, et al.: Star-convex Polyhedra for 3D Object Detection and Segmentation in Microscopy. 2020 IEEE Winter Conference on Applications of Computer Vision (WACV); 2020 1–5 March 2020. 2020; 3655–3662. Reference Source\n\nStringer C, Michaelos M, Pachitariu M: Cellpose: a generalist algorithm for cellular segmentation. bioRxiv. 2020; 2020.2002.2002.931238. Publisher Full Text\n\nWinfree S, Khan S, Micanovic R, et al.: Quantitative Three-Dimensional Tissue Cytometry to Study Kidney Tissue and Resident Immune Cells. J Am Soc Nephrol. 2017; 28(7): 2108–2118. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchindelin J, Arganda-Carreras I, Frise E, et al.: Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012; 9(7): 676–682. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHahsler M, Piekenbrock M, Doran D: dbscan: Fast Density-Based Clustering with R. J Stat Softw. 2019; 91(1): 30. Publisher Full Text\n\nWagner C, Bonnardel J, Da Silva C, et al.: Differentiation Paths of Peyer’s Patch LysoDCs Are Linked to Sampling Site Positioning, Migration, and T Cell Priming. Cell Rep. 2020; 31(1): 107479. PubMed Abstract \| Publisher Full Text\n\nStoltzfus CR, Filipek J, Gern BH, et al.: CytoMAP: A Spatial Analysis Toolbox Reveals Features of Myeloid Cell Organization in Lymphoid Tissues. Cell Rep. 2020; 31(3): 107523. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nelodiegermani, matfallet: elodiegermani/SAPHIR: A Shiny application for analyzing tissue section images (Version v1.0.0). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.4088899" }	[ { "id": "73741", "date": "11 Nov 2020", "name": "Trang Huyen Lai", "expertise": [ "Reviewer Expertise image-analysis", "genomics", "adipocyte-differentiation", "autophagy" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this article, the authors describe a shiny app to load and explore the segmentation output of fluorescence images from ImageJ/Fiji. The app provides an interactive interface to filter, select, and annotate the regions of interest as defined by the ImageJ/Fiji macros. Besides, the app allows for performing statistical testing on the selected regions. The manuscript describes the motivation for developing the app, the basic functionality, and a use case. In the use case, the app was used to filter proliferative T and B cells based on cell membranes staining and determine that more T cells interacted with phagocytes in Peyer's patches images. The article is written in clear and concise language. The motivation for developing the app and the description of the operation is sound. A few issues remain:\nMajor Issues\nThe implementation section focuses on describing the interface but doesn't explain much about the inner working of the app or the development process. (related, question 3).\n\nFour types of files are needed to use the app: Images, Data files, ROIs Files, and Legend files. Each file needs to be processed and prepared to match the formats and requirements of the app. The user has to perform many steps, including using ImageJ and R, to process the files to be able to use SAPHIR. Besides, if the user wants to combine 2 images, he or she has to prepare 4 files for each image. These complicated steps could limit the replication of the analysis by others. It is better if the providers could explain the steps and the tools that they have used to prepare their files. Or if there is an option in SAPHIR that we can use to directly prepare the files.\n\nThe article explains well using the app to perform different filtering and selection on an example image. However, it doesn't say much about the interpretation of the outputs, or the context in which these decisions could be made. (related, question 4)\n\nThe use case is very brief and it seems like many of the analysis steps were done outside of the app using custom macros. This makes it hard to evaluate the use case or determining how the app contributed to the final calculations. The authors could provide a more complete description of the use case starting from the raw images and describe how the macros work. It would be helpful to provide links to the data and the tools they used as well.\n\nThe two examples provided in the article use a single image. To be able to make any claims on the performance (related, question 5), the authors might need to perform testing with multiple images or provide a comparison with existing tools.\nMinor Issues\nSeveral screenshots from the app were included in the manuscript as figures. It might be helpful to change the size/resolution of the images for clarity. Also, highlighting the important parts of the image would make it easier to understand the interface.\n\nThe app depends on ImageJ so it should be bundled with the app or explain how to obtain and add to the app.\n\nRelated. Trying to run the app using rstudio server on rocker/verse failed. A dependency libpoppler-cpp-dev was required but not declared.\n\nProvide a viable demo, for example using shinyapps.io or rstudio.cloud would make it easier to evaluate the tool and lowers the entry cost for users.\n\nThe app is written as one long R script. This makes it very difficult to examine, modify, or extend the code. Factoring the code or making it more modular would be helpful.\n\nAfter installing the most recent versions of the required packages, trying to run the app in a fresh rstudio session through an error related to shinyjs::extendShinyjs. I was able to get it to run by adding functions = 'winprint' as an argument. Source\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Partly\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "6489", "date": "08 Apr 2021", "name": "Elodie GERMANI", "role": "Author Response", "response": "Dear reviewer, we would like to thank you for all your comments on our app and article that helped us improve them. We studied every remark and wanted to give you an answer : Major Issues 1. The implementation section focuses on describing the interface but doesn't explain much about the inner working of the app or the development process. (related, question 3). Following the recommendation of the reviewer we have now explain the development process of the app in the operation section. 2. Four types of files are needed to use the app: Images, Data files, ROIs Files, and Legend files. Each file needs to be processed and prepared to match the formats and requirements of the app. The user has to perform many steps, including using ImageJ and R, to process the files to be able to use SAPHIR. Besides, if the user wants to combine 2 images, he or she has to prepare 4 files for each image. These complicated steps could limit the replication of the analysis by others. It is better if the providers could explain the steps and the tools that they have used to prepare their files. Or if there is an option in SAPHIR that we can use to directly prepare the files. We thank the reviewer for this suggestion. We have now added in the data requirement section a description of the segmentation process. A segmentation menu to is available in the application where Fiji and the two macros used in the manuscript can be launched. 3. The article explains well using the app to perform different filtering and selection on an example image. However, it doesn't say much about the interpretation of the outputs, or the context in which these decisions could be made. (related, question 4) We agree with the reviewer and based on the use case we now describe the results we could obtain and the conclusions we were able to draw using SAPHIR on a specific application, which is the immune response initiation in murine Peyer’s patches. We also show how useful the filtering tool is to focus on a given population of cells (i.e., in our case proliferative cells of the ROI) and how interaction between the image and the scatter plot can be used to focus on a given cell of interest and to control that analysis is correct. 4. The use case is very brief and it seems like many of the analysis steps were done outside of the app using custom macros. This makes it hard to evaluate the use case or determining how the app contributed to the final calculations. The authors could provide a more complete description of the use case starting from the raw images and describe how the macros work. It would be helpful to provide links to the data and the tools they used as well. We followed the recommendation of the reviewer and have extended the section dedicated to the use case (see answer to point 3). However, since the segmentation process is now described in detail in the data requirement section, we only briefly described this step to rather focus on the contribution of SAPHIR to quantitative image analysis. As explained in the main text, all data and macros are available on GitHub in the Demonstration Files. 5. The two examples provided in the article use a single image. To be able to make any claims on the performance (related, question 5), the authors might need to perform testing with multiple images or provide a comparison with existing tools. The main goal of this manuscript was to provide a brief description of the application and its usefulness using two types of image as demos. It is now applied in the lab to study biological processes using dozens of images to validate statistics and its use will be included in several studies, but it was not the aim of this manuscript to already provide this kind of analysis. We indeed tested other tools before developing SAPHIR, such as VTEA and FlowJo, but none was really adapted to our needs as we especially wanted to have a strong interconnection between images and scatterplots. Minor Issues 1. Several screenshots from the app were included in the manuscript as figures. It might be helpful to change the size/resolution of the images for clarity. Also, highlighting the important parts of the image would make it easier to understand the interface. The resolution of the figures has been improved and Figure 6 has been modified to allow better visualization of specific parts of the panels. Moreover, the app tools now include options to adjust the cell contour thickness in the image and the size of the dots in the scatterplots. 2. The app depends on ImageJ so it should be bundled with the app or explain how to obtain and add to the app. Link to Fiji is now provided in the segmentation menu of the app, where Fiji and the two macros used in the manuscript can be launched and applied to any image. 3. Related. Trying to run the app using rstudio server on rocker/verse failed. A dependency libpoppler-cpp-dev was required but not declared. The dependency to lipoppler-cpp-dev is due to the use of the magick package, which is required to use the application. We also had this problem when trying to deploy our app on shinyapps.io. This problem can be solved using a Unix operating system instead of Windows. We have created a Docker file to use the app without this kind of issues. 4. Provide a viable demo, for example using shinyapps.io or rstudio.cloud would make it easier to evaluate the tool and lowers the entry cost for users. We are soon going to deploy a demo version on shinyapps.io. However, it will only be operable on our demo images and the user will not be able to change the images to use. 5. The app is written as one long R script. This makes it very difficult to examine, modify, or extend the code. Factoring the code or making it more modular would be helpful. We agree with the reviewer and we are going to modularize the code in the following months. 6. After installing the most recent versions of the required packages, trying to run the app in a fresh rstudio session through an error related to shinyjs::extendShinyjs. I was able to get it to run by adding functions = 'winprint' as an argument. Source We modified the script in order to correct this. Indeed, the package shinyjs was recently updated and a new argument in the function “extendShinyjs” was made mandatory. We added this modification to the latest version of our script. We hope these responses and the new version of our article will satisfy you." } ] }, { "id": "73742", "date": "30 Nov 2020", "name": "Seth Winfree", "expertise": [ "Reviewer Expertise light microscopy", "multispectral microscopy", "image analysis", "tissue cytometry", "kidney immunology", "neutrophil biology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSAPHIR is an R based tool for image cytometry. SAPHIR provides a novel R based Shinny application for analyzing an existing segmentation, an associated image and measured features of the segmented objects. The tool is intended to meet an unmet need for a flow cytometry-like exploration tool that enables 1) the highlighting of cells-of-interest in the original image volume defined by cells gated on staining intensity or morphological features and 2) the plotting of cells to a scatterplot of staining intensity or morphological features based on an image region-of-interest (ROI).\n\nMajor criticisms:\nRationale for tool development: A number of tools previously described or referenced in this manuscript include functionality similar to or overlapping with SAPHIR albeit with different implementations. Although the rationale presented by the authors is clearly explained, it is unclear what the rationale is for a new implementation of these functionalities in R (versus CellAnalyst in Python and CodexMAV and Volumetric Tissue Exploration and Analysis in Java). A tool in R could prove very useful especially in light of major R-based projects in transcriptomic analyses and data visualization and the platform's unrivaled strength in statistical analysis, but this is not addressed. Furthermore, R's strengths and weaknesses in imaging (versus other platforms) are not considered or addressed. These points require further justification and discussion.\n\nRationale for tool development: It is unclear if this tool is designed as a generalizable solution or intended only for ImageJ/FIJI users as it is tied to ImageJ/FIJI functionality to generate a number of input files-especially the segmentation which is based on the ImageJ ROI class. Although there may be a path or utility for using ROIs from Icy, CellProfiler, Ilastik or other common segmentation tools, this is not discussed and needs to be clarified.\n\nInterpretation of output results and tool functionality: The authors mention the importance of supporting multiplexed fluorescence images in the introduction, “more than 10 channel measurements”, but in neither the results nor supporting material is it clear to what degree SAPHIR supports these kinds of datasets (e.g. Lelouard et al. the manuscript). Furthermore, the support for 3D datasets is unclear, as ImageJ ROIs are limited to 2D and the link referenced in the supporting material for the “Complex Image Analysis”, https://zenodo.org/record/4056001#.X8HVRC2ZM_W, suggests the scripts used to generate the input files only consider a single optical slice. Please clarify these functionalities.\n\nTool performance: The claim of the application’s quickness is hard to assess. Although, SAPHIR appears to function well with the demonstration datasets the authors also qualify their quickness claim: “Interactivity between scatterplot and image is optional to avoid slow interaction when high-resolution images are used. In the latter case, we recommend users to perform their gating before allowing their selection to be shown in the image by ticking the appropriate box.” This apparent contradiction is unqualified, beyond “high-resolution”, the claim of quickness is poorly clarified. Please elaborate, and demonstrate if necessary, this limitation-for instance with larger or more complex datasets.\n\nManuscript: Figures are difficult to interpret due to size and resolution, especially figure 6 in which the “orange” color could not be easily identified nor the “magenta contour”. This is critical to demonstrating the behaviors of SAPHIR. Please adjust to facilitate the interpretation of the results.\n\nMinor criticisms:\nTool performance: One of the major goals appears to be addressing “bias and misinterpretation” but this is not directly discussed in the results. It is unclear in what way(s) SAPHIR directly addresses bias.\n\nTool performance: The title and introduction suggest that large or mesoscale datasets that can be generated by modern imaging modalities are a unique challenge due to their size and complexity. It is unclear to what degree SAPHIR is able to analyze datasets with a large number of cells. This is an important qualification that is not directly addressed.\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "6490", "date": "08 Apr 2021", "name": "Elodie GERMANI", "role": "Author Response", "response": "Dear reviewer, we would like to thank you for all your comments on our app and article that helped us improve them. We studied every remark and wanted to give you an answer : Major criticisms: 1. Rationale for tool development: A number of tools previously described or referenced in this manuscript include functionality similar to or overlapping with SAPHIR albeit with different implementations. Although the rationale presented by the authors is clearly explained, it is unclear what the rationale is for a new implementation of these functionalities in R (versus CellAnalyst in Python and CodexMAV and Volumetric Tissue Exploration and Analysis in Java). A tool in R could prove very useful especially in light of major R-based projects in transcriptomic analyses and data visualization and the platform's unrivaled strength in statistical analysis, but this is not addressed. Furthermore, R's strengths and weaknesses in imaging (versus other platforms) are not considered or addressed. These points require further justification and discussion. Due to the short format chosen for our manuscript (allowed initial length of 1000 words), it was not possible to include a discussion on the advantage/disadvantage of each software and its environment. CodexMAV is a commercial software but available for free as an ImageJ plugin. It needs FCS files to run. The software is very powerful to do spatial analysis (t-SNE) of cell populations in tissues, but we didn’t test it in our datasets, yet. CellProfiler (Analyst) is very interesting in term of segmentation and analysis but as far as we know, scatterplots and images are not interconnected. VTEA is also a great and easy application to use, as it is an ImageJ plugin. However, at the time we needed quantitative analysis to be done, segmentation was included in VTEA without all the flexibility that was required for the segmentation of our datasets. We therefore asked the VTEA developer to add the possibility to load ROIs from ImageJ, which is done now. However, the strong interconnection between the image and the scatterplot that we wanted to have to control and correct cells identity was lacking. We have added a sentence in section Operation to explain why we have chosen R for developing SAPHIR application. 2. Rationale for tool development: It is unclear if this tool is designed as a generalizable solution or intended only for ImageJ/FIJI users as it is tied to ImageJ/FIJI functionality to generate a number of input files-especially the segmentation which is based on the ImageJ ROI class. Although there may be a path or utility for using ROIs from Icy, CellProfiler, Ilastik or other common segmentation tools, this is not discussed and needs to be clarified. Following reviewer recommendation, we have now added a sentence in the data requirement section to help users with the conversion of COI obtained from other software into the Fiji .roi format. 3. Interpretation of output results and tool functionality: The authors mention the importance of supporting multiplexed fluorescence images in the introduction, “more than 10 channel measurements”, but in neither the results nor supporting material is it clear to what degree SAPHIR supports these kinds of datasets (e.g. Lelouard et al. the manuscript). Furthermore, the support for 3D datasets is unclear, as ImageJ ROIs are limited to 2D and the link referenced in the supporting material for the “Complex Image Analysis”, https://zenodo.org/record/4056001#.X8HVRC2ZM_W, suggests the scripts used to generate the input files only consider a single optical slice. Please clarify these functionalities. SAPHIR is supporting multichannel images and Z stack image (ijtiff is used to load the image). We indeed used 3D images but following 3D segmentation only the main COI corresponding to the largest nucleus area was kept for calculating the intensity of other channels. In Fiji, the ROI are linked to specific slices allowing this operation. Thus, we used 2D ROI of 3D images for SAPHIR analysis. This is now explained in the data requirement section. Of note, it is now possible to obtain 3D ROI in Fiji using a Plugin from Thomas Boudier (https://imagejdocu.tudor.lu/plugin/stacks/3d_roi_manager/start#d_roi_manager). 4. Tool performance: The claim of the application’s quickness is hard to assess. Although, SAPHIR appears to function well with the demonstration datasets the authors also qualify their quickness claim: “Interactivity between scatterplot and image is optional to avoid slow interaction when high-resolution images are used. In the latter case, we recommend users to perform their gating before allowing their selection to be shown in the image by ticking the appropriate box.” This apparent contradiction is unqualified, beyond “high-resolution”, the claim of quickness is poorly clarified. Please elaborate, and demonstrate if necessary, this limitation-for instance with larger or more complex datasets. As the application could be slowed down with images >300MB, we decided to remove the term \"quickly” from the abstract of the manuscript. Moreover, the limitation of 300MB is now specified in the implementation section. Big images (with multiple slices and channels) have to be stored on SSD drive to limit slow interaction. Since our application was mainly used for tissue section < 20 µm, we did not test datasets > 300MB, yet. 5. Manuscript: Figures are difficult to interpret due to size and resolution, especially figure 6 in which the “orange” color could not be easily identified nor the “magenta contour”. This is critical to demonstrating the behaviors of SAPHIR. Please adjust to facilitate the interpretation of the results. The resolution of the figures has been improved and Figure 6 has been modified to allow better visualization of specific parts of the panels. Moreover, the app tools now include options to adjust the cell contour thickness in the image and the spot size in the scatterplot. Minor criticisms: 1. Tool performance: One of the major goals appears to be addressing “bias and misinterpretation” but this is not directly discussed in the results. It is unclear in what way(s) SAPHIR directly addresses bias. By allowing to analyze for each cell of the scatterplot its position and its characteristics in the image, SAPHIR allows to check that the parameters obtained automatically with the segmentation and other analysis algorithms fit with the observation of the selected cells in the image. Then, the annotation menu allows to correct any bad analysis (e.g., cell identity). We have now extended the use case section to better describe SAPHIR use. 2. Tool performance: The title and introduction suggest that large or mesoscale datasets that can be generated by modern imaging modalities are a unique challenge due to their size and complexity. It is unclear to what degree SAPHIR is able to analyze datasets with a large number of cells. This is an important qualification that is not directly addressed. Our aim was clearly not to analyze mesoscale datasets. We apologize if that is the impression given by our title or introduction, although we see no allusion to it in the title and only a small reference in the introduction (i.e., “10 µm to more than 200 µm when clearing methods are used”) but not related to the description of the application. Actually, as mentioned in the introduction our main goal was to obtain an interconnection between the representation of image parameters in scatterplot and the image itself in order to analyze positioning of scatterplot-selected cells in the image and to check and correct any analysis errors by direct observation of the selected cells. We hope these responses and the new version of our article will satisfy you." } ] } ]	1	https://f1000research.com/articles/9-1276
https://f1000research.com/articles/8-37/v1	09 Jan 19	{ "type": "Case Report", "title": "Case Report: A rare cause of vaginal bleeding at Keren Zonal Referral Hospital, Eritrea", "authors": [ "Million Abraha", "Shiden Solomon", "Shiden Solomon" ], "abstract": "Vaginal bleeding due to leech infestation is a very uncommon but important gynaecological problem. This report presents the case of a 65-year-old woman who presented to Keren Regional Referral Hospital, Eritrea, with vaginal bleeding of two and half weeks duration, dizziness and fatigue. On the day of her admission complete blood cell count and speculum exam were done and she was diagnosed with anaemia due to cervical leech infestation. Routine speculum exam for vaginal bleeding is recommended in cases with history of holy water or springs visits to prevent unnecessary diagnostic tests and for rapid management.", "keywords": [ "Leech", "Vaginal bleeding", "Anemia", "Infestation", "Case report" ], "content": "Introduction\n\nLeeches, which belong to the phylum Annelida and class Hirudinea, are blood-sucking parasites of most mammals, including humans. Though there are about 650 known species of leech, only a few of them are a threat to human health. People and livestock that walk close by and frequently have access to marsh areas or slow-moving streams and brocks are among the frequent victims of leech infestation1. Moreover, there have been reports of climatic variation associated with leech infestation, where leeches are more frequently found in tropical and subtropical areas2.\n\nLeeches have a long slender body, 5-45mm, with an oral sucker as a mouth and caudal end for movement3. Leeches bite different body sites of victims, which includes the pharynx, larynx, oesophagus, rectum, vagina, urethra and bladder, which have been reported across different publications1,3,4.\n\nWhen leeches become in close contact with human bodies, they tend to attach themselves to the mucosal surfaces and secrete an anticoagulant that leads to excessive bleeding from the attachment site2,5. If it is not diagnosed as early as possible, this seemingly harmless parasite, could lead to some life threatening anaemia and shock2. In this paper we present a referral case of rare vaginal bleeding due to cervical leech infestation.\n\n\nCase presentation\n\nA 65-year-old para IV mother with no history of abortion, presented with vaginal bleeding of 17 days duration. She was referred to Keren Zonal Referral Hospital, Eritrea, from Adi-tekelezan Health Centre with a diagnosis of vaginitis.\n\nUpon arrival, the general condition of the mother was stable. She had a fresh blood soaking her clothes. The patient reported that she was bleeding intermittently at home followed by big clots of blood. Since the start of the bleeding the patient was feeling dizzy and was becoming fatigued easily. A few days prior to admission to hospital, she was feeling an abnormal moving sensation in her abdomen. The patient stated that previous to her symptoms, she had visited Amne-Tekle Bahri, a holy water nearby, to help her alleviate headache and psychological problems.\n\nUpon thorough examination, the patient had blood pressure of 90/40 mm Hg (normal range 90/60 mm Hg to 120/80 mm Hg), pulse rate 140 beats per minute (normal range, 60 to 100 beats per minute) and respiratory rate 33 per minute (normal range, 12 to 18 breaths per minute). She had pale conjunctiva. The perineal area was stained with blood, the cervix was closed, and the uterus was normal in size and consistency. Speculum examination revealed an actively moving worm attached to the external orifice of the cervix (a similar visual representation can be seen in Tilahun (2015)6 Figure 1). Complete blood count showed a haemoglobin level of 4.6g/dl (normal range, 12 g/dL to 16.0 g/dL) and platelet count of 110,000 cells/microliter (normal range, 150 x 103 μL to 400 x 103 μL). Abdominal ultrasound finding was normal.\n\nThe patient was transfused with two units of blood immediately and the bleeding site was gently washed with saline and a bit of alcohol to facilitate the detachment of the leech. With a lot of care, the leech was removed gently using sterile forceps. After stabilising the vital signs (respiratory rate 17 breaths per minute, blood pressure 110/80 mm Hg, pulse rate 80 beats per minute), the patient was sent home after four days with iron tablets and with advice to take a good preventive measures if she had ever to go to the holy water again. She was also informed to bring the accompanying family members who went with her if they showed any unusual symptoms. Pre-discharge complete blood cell count showed an increased Hg level of 8g/dl and platelet count of 120,000 cells/microliter. The patient was asked to come back after two weeks for follow-up.\n\n\nDiscussion\n\nVaginal bleeding due to leech infestation is a very uncommon complaint in women visiting gynaecological and obstetrics departments7–9. Vaginal bleeding due to worm infestation could usually be missed if thorough investigations and physical examination are not done accordingly. A speculum exam should be done to see the source of bleeding and possible leeches before entertaining other differential diagnosis, especially if a patient reports visiting water bodies. Great care should be taken in removing the worm from its attachment. It should not be forcefully removed as the jaws of the worm might remain at the bite site which might cause additional bleeding or lead to later malignancies10. Preventive measures such as self-checking after washing in ponds and waterfalls are necessary to minimize infestation and instructions should be given as part of routine health education. An awareness program should be initiated to inform clinicians working in clinics near such sources of water.\n\n\nConsent\n\nWritten informed consent for the publication of the case report was obtained from the patient.\n\n\nData availability\n\nNo data is associated with this article.", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAuthor information\n\nMA was responsible for the gynaecological management of the patient while based at Keren Zonal Referral Hospital in 2012. SS was the nurse collaborating on this case while based at Halibet Hospital.\n\n\nReferences\n\nDeka PM, Rajeev TP: Unusual cause of hematuria. Urol Int. 2001; 66(1): 41–42. PubMed Abstract \| Publisher Full Text\n\nIynen I, Sogut O, San I, et al.: A Rare Cause of Epistaxis, Hemoptysis and Anemia: Leech in the Nasopharynx. Journal of Medical Cases. 2010. Publisher Full Text\n\nSaha PK, Roy S, Bhattacharya D, et al.: Leech bite: a rare gynecologic emergency. MedGenMed. 2005; 7(4): 73. PubMed Abstract \| Free Full Text\n\nRaj SM, Radzi M, Tee MH: Severe rectal bleeding due to leech bite. Am J Gastroenterol. 2000; 95(6): 1607. PubMed Abstract \| Publisher Full Text\n\nChin TH: Further note on leech infestation in man. J Parasitol. 1949; 35(2): 215. PubMed Abstract\n\nTilahun T: Vaginal Leech Infestation: A Rare Cause of Hypovolumic Shock In Postmenopausal Woman. Ethiop J Health Sci. 2015; 25(4): 377–380. PubMed Abstract \| Free Full Text\n\nAribarg A, Phupong V: Vaginal bleeding in young children. Southeast Asian J Trop Med Public Health. 2003; 34(1): 208–212. PubMed Abstract\n\nHailemariam B: Post menopausal vaginal bleeding due to vaginal wall leech infestation. Ethiop Med J. 1995; 33(3): 183–185. PubMed Abstract\n\nPrasad SB, Sinha MR: Vaginal bleeding due to leech. Postgrad Med J. 1983; 59(690): 272. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShamsaddini S, Dabiri S: Basal cell carcinoma on nickel dermatitis after leech applying. East Mediterr Health J. 2000; 6(1): 197–199. PubMed Abstract" }	[ { "id": "51366", "date": "31 Jul 2019", "name": "Temesgen Tilahun", "expertise": [ "Reviewer Expertise Gynecology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAbstract\nLine 5 ‘complete blood cell count’ write as 'complete blood count'.\nCase presentation\nGood to replace the word ‘mother’ with ‘patient’.\n\nLine 2 ‘17 days duration’ write as ‘17 days’.\n\n‘a bit of alcohol to facilitate the detachment of the leech’. It is clear that alcohol isan irritant to the vagina. How did you use it? Is that contraindicated in the vagina? This needs explanation.\n\nThis patient was appointed for follow up but what happened then?\n\nThe authors must include a picture of the leech.\nDiscussion\n‘Great care should be taken in removing the worm from its attachment’.What does great care mean? Rather it is good if you recommend other techniques.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [ { "c_id": "6537", "date": "08 Apr 2021", "name": "Million Abraha", "role": "Author Response", "response": "Generally there were a minor changes required in the whole manuscript. In the abstract section I removed the word ‘count’ in ‘complete blood cell count’. In the case presentation section the word mother was replaced with patient and the days duration with just 17 days. In our practice we usually apply alcohol before we remove the leech from its attachment site. Although alcohol can irritate the wounded mucosal surface, it can still help you in removing the leech as it irritate the leech at the same well. To best of our knowledge we still did not find if this contradicting to the idea that no alcohol should be used in removal of leechs. The patient was appointed for follow up and the wounded site has healed and there was no fresh blood seen from the previous attachment site. We did not take a photo of the leech because initially there was no idea to publish it as the procedure was being done. In discussion section, ‘Great care should be taken in removing the worm from its attachment’. This means we tried to be meticulous with the procedure as it might hurt other part of the vagina during the process. I hope i this was explanatory to the questions raised. Thank you everyone for their inputs." } ] }, { "id": "52696", "date": "20 Aug 2019", "name": "Hossein Zarrinfar", "expertise": [ "Reviewer Expertise Microbiology", "Mycology." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the manuscript entitled \"Case Report: A rare cause of vaginal bleeding at Keren Zonal Referral Hospital, Eritrea\", the authors have described vaginal bleeding. This manuscript is interesting and educational, but there is no figure related to the leech, that should be added. By the way, there are some grammatical and spelling errors in English too that should be corrected by an expert.\n\nAbstract:\n“This report presents the case of a 65-year-old woman who presented to Keren Regional Referral Hospital, Eritrea, with vaginal bleeding of two and half weeks duration, dizziness and fatigue. On the day of her admission complete blood cell count and speculum exam were done and she was diagnosed with anaemia due to cervical leech infestation”. The sentences are unclear and need revision.\n\nThe conclusion should be revised according to study aim again. Hence, “Routine speculum exam for vaginal bleeding is recommended in cases with history of holy water or springs visits to prevent unnecessary diagnostic tests and for rapid management” to be omitted.\nIntroduction:\n“…more frequently found in tropical and subtropical areas”, this sentence needs another newer reference. Please add this1.\n\n“If it is not diagnosed as early …. Anaemia and shock” the sentences are unclear and needs a revision.\nDiscussion:\nThis section should be revised as predisposing factors to leeches in other studies. The conclusion should be revised according to study aim and the results again.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-37
https://f1000research.com/articles/10-276/v1	06 Apr 21	{ "type": "Research Article", "title": "Summary of two questionnaires designed to understand the research climate for Bioimage Analysts in the UK between 2016-2019", "authors": [ "Dominic Waithe" ], "abstract": "Background: Bioimage analysis is an emerging field within the global research community. It is an interdisciplinary discipline which requires knowledge of biology, image analysis and biophysics. This report represents the analysis and discussion of two questionnaires run by the Image Analysis Focused Interest Group of the Royal Microscopical Society (IAFIG-RMS). The goal of this document, which represents the analysis and interpretation of these questionnaires, is to highlight the current research climate for Bioimage Analysts in the UK and discusses some of the problems and possibilities for this emerging discipline. Methods: Two questionnaires (2016 and 2019) were developed and sent to researchers in the UK using mailing lists and forums specific for microscopy and image analysis. The participants were asked a range of questions spanning different aspects of their work and funding. Respondents were collected and analysed using Jupyter notebooks. Results: The analysis of the responses from these questionnaires highlighted many interesting issues and aspects of this community. It is clear that a major issue for the community is the nature of the funding and the long-term career possibilities available. Furthermore, the issue of independence is discussed with clear evidence that researchers would like to pursue their own research with the option of dedicated time to support the research of others. Conclusions: It is our hope that this study will help catalyse funding opportunities which help support this emerging discipline and help it establish a unique identity for itself within the research community in the UK and beyond.", "keywords": [ "bioimage analysis", "community", "questionnaire", "career" ], "content": "Introduction\n\nA Bioimage Analyst is a research scientist who performs, as their primary role, image analysis in the context of biomedical research. This broadly involves developing pipelines of analysis which can be used to extract data from images acquired from microscopy. This role is discrete from general image analysis or computer vision due to the close integration it has with biological and biophysical research practises. Schemes like Neubias have done an excellent job of highlighting the need for Bioimage Analysts and have also provided good training opportunities for them1,2 across the EU and beyond. Unfortunately, however, the Neubias EU Cost funded scheme has now ended and although multiple aspects of the scheme will continue, it is up to individual countries, universities and funders to develop this movement further. This support includes providing career opportunities in universities which support this role (e.g. lectureships, professorships, facility roles) and also for the research bodies to fund these roles at multiple levels (e.g. fellowships, core funding, project grants).\n\nIn the UK research bodies and universities in the UK have not yet addressed the unique nature of this group of people and often researchers feel their careers have been side-lined as a result. To address this, two questionnaires were designed and distributed. The initial questionnaire was centred on the theme \"Do you use or develop bioimage analysis for life sciences research?\". In the second questionnaire we then focused on the issue of funding within the community of bioimage analysis and drill down further into this subject. In summary, we show that there is an active community of scientists who are performing image analysis in terms of their own research and for the benefit of others. The following document discusses the points raised.\n\n\nMethods\n\nThe questionnaires were distributed through mailing lists specific to microscopy and image analysis, and addressed to the UK readership of these lists. Questionnaire 1 was distributed to the following mailing lists (on the specified dates): ImageJ mailing list (IMAGEJ@LIST.NIH.GOV)(23/09/2016), UK-Eurobioimaging-project (uk-eurobioimaging-project@jiscmail.ac.uk) (23/09/2016), Confocal Microscopy mailing list (LISTSERV@LISTS.UMN.EDU)(23/09/2016), Royal Software Engineers (everyone@rse.ac.uk) (23/09/2016). An additional email was sent to the Image World mailing list (sci-diku-imageworld@list.ku.dk) (29/09/2016). No follow-up emails were sent. 89 of the 99 respondents had submitted official UK academic email addresses. No submissions were excluded. In questionnaire 1 a Wordcloud (1) was created from the job title entries using the website: https://www.wordclouds.co.uk/. using an online resource For Questionnaire 2 a UK specific microscopy image analysis mailing list was available (IAFIG Jiscmail) and used to distribute the emails: Two emails were sent to this list (IAFIG-RMS@jiscmail.ac.uk) (10/01/2019 and 16/01/2019). In addition, the questionnaire was promoted through a tweet on Twitter (community response: 22 Retweets, 6 Tweets, 13 Likes), and also with a post on the Image.sc image analysis web forum (https://forum.image.sc). Furthermore an email was forwarded to the UK-Eurobioimaging-project (uk-eurobioimaging-project@jiscmail.ac.uk)(11/01/2019). 65 of the 84 respondents submitted official UK academic email addresses. No submissions were excluded. 24 of the original 99 participants of Questionnaire 1 returned and answered Questionnaire 2.\n\nBoth questionnaires were designed and run using the website SurveyMonkey (https://www.surveymonkey.co.uk).\n\nPlease refer to the Extended data3 for details of the questions asked for each Questionnaire. Responses were analysed using the scripting language Python (3.7.4) and were analysed and visualised using Jupyter lab notebooks.\n\nDue to the non-sensitive nature of the content, in addition to the lack of identifying information being included in this publication, no ethical approval was sought for this study. None of the data presented here, or in the accompanying Zenodo repository, can be used to identify survey participants, and the data is not accessible through the SurveyMonkey platform used to host the questionnaires (https://www.surveymonkey.com/mp/policy/privacy-policy/). Completion of the questionnaire after reading the information sheet was taken as consent to participate in the survey.\n\n\nResults and discussion\n\nBoth questionnaires have proved highly informative.\n\nMany researchers who identify as being Bioimage Analysts have developed the necessary expertise for themselves having responded to a need in the community. As such they often come from a diverse range of roles and are classified according to their employers in a variety of different ways. When we asked scientists who perform bioimage analysis, \"What is your current job/position title?\" we got a very broad distribution of answers.\n\nQ1.1: What is your current job/position title? Out of the 99 people that answered the first questionnaire only a maximum of three people gave the same answer to the above question and very few people gave the answer ‘Image Analyst‘ and no-one gave the answer ‘Bioimage Analyst‘. The above ‘Word Cloud Image‘ (Figure 1) gives an idea of the range of nomenclature for individuals in this discipline. Going by their job title it suggests that bioimage analysis is not the main focus of their work.\n\nParticipants were able to submit one or more words describing their job position/title. Each word was treated independently.\n\nQ1.2: Do you feel that your current job/position title accurately describes the work that you do? In Figure 2 we show the outcome from asking: \"Do you feel that your current job/position title accurately describes the work that you do?\" To this question 50.5% of individuals said ’YES’ to the above question whereas around 43.4% said ’Not completely’ and 6.1% said ’No’, not at all. This suggests that there is a large component of individuals for whom their job title and description doesn’t quite describe the focus of their work. This doesn’t prove that they should be named ’Image Analyst’ or similar but it does suggest individuals are assuming or taking on roles which are not reflected by their job-title. As we see later, there is evidence to suggest these individuals are mostly performing image analysis.\n\nParticipants were able to select only one answer.\n\nQ1.3: Which of the following categories best describes your working relationship with image analysis? We posed the question, \"Which of the following categories best describes your working relationship with image analysis?\". The responses to this question were very interesting (Figure 3). In total, 35.4% saw themselves as ’Image Analysis Users’. These are individuals who treat image analysis as a tool that they use in their research. Although potentially skilled, they do not create or develop analysis methods, algorithms or pipelines. Many of these researchers are likely laboratory bioscientists interested in using image analysis tools for testing biological hypotheses, without an active role in developing algorithms and pipelines. The remaining 64.6% of individuals put themselves in a more specialist role, mostly either as a Bioimage Analyst, Specialist Image Analyst but a few used the term Software Engineers. This essentially means that 64.6% of the individuals who filled out this questionnaire have a professional level of image analysis expertise and are employed in the life sciences. Along with the first two questions, it suggests that these individuals in their roles are not defined according to the actual job they are performing, but have found themselves in these roles as means to do what they want, or what is needed (i.e. image analysis). In our follow-up questionnaire (section 2, Figure 7 and Figure 8) we asked the same question but provided more focused answers and analysis.\n\nParticipants were able to select only one answer.\n\nQ1.4: Often we perform different roles at different times. how much of your time, on average, do you commit to the following activities? For this question subjects were asked how they organise their day-to-day research between the activities of a Bioimage Analyst, Software Engineer, Image Analysis User and other research activities. We weighted people’s answers depending on whether they stated that they spent all of their time (weight = 8), most of their time (6), half of their time (4), just some (1) or none of their time (0) performing the specified activities. Broadly speaking most individuals spent most of their time performing some kind of image analysis, whilst compacting all other activities into a smaller proportion of their time (Figure 4). This is very interesting as it shows that most of the individuals who filled out this questionnaire are indeed Image Analysts and that other forms of research and experimentation are secondary to their data analysis.\n\nParticipants were able to select multiple answers.\n\nQ1.5: If you are interested in pursuing image analysis as potential career, do you feel as though there are sufficient options for career progression in your chosen field of academia? Figure 5 shows the distribution to answers from the following question: \"If you are interested in pursuing image analysis as potential career, do you feel as though there are sufficient options for career progression in your chosen field of academia?\" The distribution of answers to this question is very clear. Of those individuals interested in pursuing image analysis 63.4% of them said that they did not have sufficient career options to develop in this direction. This essentially means that although these individuals are ful-filling an important and high-demand area of bioscience research, there is not a sufficient framework in place to support their specific career development. The previous questions prove that many individuals are performing image analysis as the dominant aspect of their day-to-day research, even if it isn’t their obvious job description.\n\nParticipants were able to select only one answer.\n\nWe reasoned that the issues affecting the bioimage community highlighted in the first questionnaire relate ultimately to the organization of funding in the UK. In our follow-up questionnaire (questionnaire 2), we asked a number of questions relating to the funding of scientists that perform some kind of bioimage analysis in their work. Through this characterization (84 respondents) we have been able to clarify the funding sources which are funding bioimage analysis and are now in a position to make suggestions to the community as a whole.\n\nQ2.3: What best describes your position? (Please choose the closest role). Q2.3 (Figure 6) was designed to find the closest job description which scientists will classify themselves into. Previously we showed the diversity of titles that people will assign to themselves in (Q1.1), but here we ask specifically which title best describes their position. At the moment, the category that is most dominant among those doing bioimage analysis is Facility Manager (31%), followed by post-docs in a research group (19%). This is to be as expected as scientists, from within a facility, will need to assist researchers by providing image analysis expertise, whereas post-docs will be performing analysis to pursue their own research or developing new techniques for the community. If we compare the ratio of facility-based staff (44.1%) to research group staff (41.5%) who are doing some form of bioimage analysis, the ratio is about 50:50. Therefore a simplified view of this is that from those that filled out questionnaire approximately 50% of scientists are on an academic career track and the other 50% can be considered research technical professionals.\n\nWhat best describes your position? Participants were able to select only one answer.\n\nQ2.4: Which of the following categories best describes your working relationship with image analysis? We asked the participants to describe their relation with image analysis (Figure 7, Figure 8) by checking the categories they most associated with the role. We took the answers and pooled those individuals whom associated mostly with facilities and compared it to those who predominantly associated with research groups as determined from Q2.3. Figure 7 and Figure 8 show that there are some clear differences between the two cohorts.\n\nParticipants were able to check multiple options.\n\nParticipants were able to check multiple options.\n\nThe main differences between the work relating to bioimage analysis between a facility and a research group is that much more training is being performed in the facility, whereas the ’specialist image analyst’ and also the ’Image Analysis Users’ are more abundant in research groups. The proportion of individuals performing training in research groups (5.6%) is low compared to those performing training in a facility (30.6%). Conversely however there are more ‘Image Analysis Users’ in research groups (facilities: 11.8%, research groups: 23.5%) and also more ‘Specialist image analysts’ (facilities: 15.3%, research groups: 23.5%). This shows that research groups are more interested in applying algorithms through software (as an Image Analysis User) and/or developing specialist algorithms and approaches. Interestingly however the amount of bioimage analysis is consistent amongst the groups (facilities: 25.9%, research groups: 23.5%) showing that connecting pipelines of algorithms is a common goal, whether it be to support users or to develop novel pipelines of analysis.\n\nQ2.5: Where does your funding come from? (0–100)% The academic positions (i.e. PhD positions, post-doc, PI positions) attract mainly funding from research grants, fellowships, with some PIs being partially funded by lectureships (Figure 9). Facility managers and research assistant positions are funded predominantly by cost-recovery and core-funding. This is fairly typical in the life sciences where facility and technical staff are mainly funded by core-funding or funding generated within an institute whereas researchers are attracting funding mainly from research grants\n\nParticipants were asked to gauge the approximate funding contributions (0–100%) of their position from the following sources: CF (Core Facility), RG (Research Grants), UL (University Lectureship), FG (Fellowship Grant), CR (External and Internal Cost Recovery), OF (Other funding). Center, 0% funding, Outside Circle, 100%, each graduation represents 20%. Values in each category were averaged over each class of participant.\n\nQ2.6: Which UK Research Councils or Charities fund your research? From the answers generated from Q2.6 it is clear the main funders for bioimage analysis at the moment are the MRC, BBSRC1, EPSRC, Wellcome and CRUK (Figure 10). This result is fairly consistent with the idea that the research grants are indirectly funding bioimage analysis research through conventional funding routes.\n\nParticipants were able to check multiple options.\n\nQ2.7: How much longer will your current funding last? (If you have multiple funding sources, choose the one that runs out first). The more academic posts (post-docs, PhDs, PI) tend to have a more narrow funding horizon, whereas the facility roles and research assistant roles can (not always) benefit from longer funding (Figure 11). This is shown by the apparent skew of distributions toward the left-hand side in the categories of Post-doc in research group, PhD in research group and PI. This highlights the known career dynamic that influences research and facility positions. Facility positions often mean your academic career is limited, but can often mean you have access to better job security, a factor which influences many researchers whom take up these posts.\n\nCategories represent: <6m (< 6 months), 1y (1 year), 2y (2 years), 3y (3 years), 4y (4 years), 5+ (5 years and more), UR (Until retirement).\n\nQ2.8: Have you found that your funding/university have included adequate resources for you to develop professionally (i.e. have been allowed and encouraged to apply for your own funding, attend conferences, network)? The answers from Q2.8 suggest that the majority (54.8%) of researchers in 2019 have sufficient money and opportunities to develop professionally (Figure 12). In total, 33.3% believe more opportunities are needed, but that there are some opportunities available for them. This is a fairly good representation of how the existing funding opportunities support career development in the life sciences through conference attendance, networking and opportunities to apply for certain kinds of grants.\n\nData was pooled from across job descriptions for this question. Participants were able to select multiple answers.\n\nQ2.9: Statement: Due to the sometimes mixed and ad-hoc nature of funding sources it is possible that one’s source of funding has been detrimental to your efforts to support your local community as a whole (i.e. you have not been able to assist people with general problems because of commitments to a particular funding source). Question. Do you agree with the above statement? We asked Q2.9 to ascertain whether participants in their current position felt as though they have enough flexibility to address the needs of their institute. There replies were broad to this question with a range of answers representing a unique set of circumstances (Figure 13). In general, most participants felt that they could assist the local community to some level of capacity with only 4.8% saying that they couldn’t address the needs of the local community, due to tight funding restrictions. This shows that once funding has been obtained then there is sufficient flexibility within the funding’s remit to support the needs of the local community.\n\nParticipants were able to select only one answer. Data was pooled from across job descriptions for this question.\n\nQ2.10: Statement: Individual researchers, in terms of their career, are feeling as though they must choose in a binary sense between pursuing independent research or a service/support role. Question: Would you be interested in funding which financially assisted you to support your local community (i.e. training and assisting in other people’s projects), but also allowed some time to pursue your own independent research interests (similar to a fellowship)? The answer to this question was very clear (Figure 14); a job role whereby you can have your own ongoing research, but also support the community directly, is seen as ideal. Most Bioimage Analysts on an academic track realise that for good uptake of their approaches they need to work directly with users and support their ongoing work. Conversely, if novel techniques are developed these should be allowed to form the genesis of academic progression, even if the work was done in support of another researcher’s research project. This mixed model of research and support would be a good definition for establishing and representing Bioimage Analysts as a discrete entity, a system which allows them to have an identity in both the academic and technical settings. In addition to the set answers, we allowed the participants to also input their own answers. These are included in the Extended data3 and echo the results from the main question.\n\nParticipants were able to select only one answer. Data was pooled from across job descriptions for this question.\n\n\nConclusions\n\nCurrently the research environment in the UK does not formally recognise Bioimage Analysts as a discrete entity, in terms of its funding opportunities, and career development. Researchers who perform this role are a mixed-bag with a variety of backgrounds and job descriptions (Q2.4). The discipline is new and so this is understandable, however it is clear that as the discipline grows, to retain top staff it is important that this discipline is adequately recognised and funded. Although there is flexibility in the system that does allow individuals who are doing bioimage analysis to exist, and train (Q2.8), it doesn’t do enough to support their long-term career goals (Q1.5).\n\nNeubias did a great job of defining what a Bioimage Analyst is, and with this definition it becomes easier for research bodies and the community to recognise and fund scientists within this role. We hope however that the Bioimage Analyst job description retains its fluidity and does not get defined purely as an academic or as a technical role. Due to the ad-hoc nature of the role up and to this point, it has allowed a certain degree of flexibility in how individuals have fulfilled this responsibility. Because someone decides to leave the academic track in favour of a role which is more aligned with support should not mean the outcomes of their work prohibit them from perhaps returning to an academic track at a later date. Quite the opposite, we want to encourage individual scientists to support the community more. The best way to encourage top-researchers to disseminate their specialist knowledge is through encouraging them with research money predicated on them also providing training and support content. We also recognise that scientists crossing from more wet-laboratory practises may want to spend time in a facility, to develop their technical skills, before going back into an academic career track. Individuals should be encouraged to do this and so metrics of productivity should include co-authorships, software maintenance and training. Therefore, we think it would a good idea to introduce fellowships which encourage a significant amount of support in the role. Furthermore, we think that facility staff should be allowed to apply for grants which mean they can offset their time from support to allow them to develop their academic independence, whether it be through maintaining software they have developed, or allowing them to pursue a research question based on an interest which has emerged from their work in the facility. The vast bulk of participants in this questionnaire agree with this sentiment of mixed research/support fellowships (Q2.10).\n\nIn summary this report has documented and summarised the results of two questionnaires designed to probe and understand the research community in the UK who are currently engaged in some form of bioimage analysis. It is essential that research bodies properly recognise this discipline and do so by providing long-term career possibilities which are suited to this role. By supporting initiatives involving bioimage analysis and also through recognising the efforts of groups like Neubias and the IAFIG, funders, universities and research bodies will yield the benefits from an emerging and highly productive community.\n\n\nData availability\n\nZenodo: Underlying data from two questionnaires used to understand the research climate of Bioimage Analysts in the UK between 2016–2019, http://doi.org/10.5281/zenodo.46054163.\n\nThis project contains the following underlying data:\n\nQuestionnaire analysis 1.ipynb (Jupyter Notebook, Python 3.5+)\n\nQuestionnaire analysis 2.ipynb (Jupyter Notebook, Python 3.5+)\n\nQuestionnaire 1+2.zip (zip file containing questionnaire 1+2 raw data (with email addresses removed))\n\nZenodo: Underlying data from two questionnaires used to understand the research climate of Bioimage Analysts in the UK between 2016–2019, http://doi.org/10.5281/zenodo.46054163.\n\nThis project contains the following extended data:\n\nExtended Data 1: Questions asked for Questionnaire 1 and 2 using SurveyMonkey (Extended Data.pdf).\n\nExtended Data 2: Questionnaire 2 respondents localized by UK (and Ireland) university affiliation. Location determined from academic email address suffix (Extended Data.pdf).\n\nExtended Data 3: The ’other’ points raised as an optional comment field to Q2.10 (Extended Data.pdf).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nWe would like to thank the following members of the bioimage analysis research community who provided proof-reading and feedback on this study: Janos Kriston-Vizi, Siân Culley, Michelle Peckham, Elisabeth Kugler. We would like to thank the Royal Microscopical Society who provided administrative support and the UKRI MRC who provided salary support for Dominic Waithe during this time.\n\nThis publication was supported by COST Action NEUBIAS (CA15124), funded by COST (European Cooperation in Science and Technology).\n\n\nFootnotes\n\n1 It should be noted that BBSRC was, through mistake, not included when the questionnaire was first released. This mistake was corrected and results pooled from mentions of \"BBSRC\" in the “Other” category. As a consequence, the BBSRC may be somewhat under-represented in this category.\n\n\nReferences\n\nMiura K: A Pilot Survey on Bioimage Analysis Needs. Results Summary. 2015.\n\nhttp://www.eubias.org/NEUBIAS/\n\nWaithe D: Underlying data from two questionnaires used to understand the research climate of Bioimage Analysts in the UK between 2016-2019. (Version 1.0) [Data set]. Zenodo. 2020. http://www.doi.org/10.5281/zenodo.4562390" }	[ { "id": "82875", "date": "12 Apr 2021", "name": "Julien Colombelli", "expertise": [ "Reviewer Expertise Light Microscopy" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe author provides a report on two surveys aimed to capture a snapshot for the status of Biomage analysis as a field of research in the UK, including the research/service activities of its stakeholders, the staff distribution and funding perspectives, and their perception on the adequacy of funding in this field.\nOverall, the results are very interesting and provide, although at the national level for UK and with a relatively contained numbers of respondents, a rather precise snapshot that no other national or international survey, to date and with data, has reported to support a discussion on the place of bioimage analysis and bioimage analysts in Life Science.\n\nIn general, the paper has a great value for the community and should stimulate other countries to perform similar studies to capture invaluable data on the progress made in Life science towards supporting Biomage Analysis, and its staff, as a critical activity that needs better definition and recognition.\nI recommend minor clarifications detailed below.\nGeneral comments on Methods:\nWere all questions optional or mandatory?\nSee below for Q1.5 where it is relevant to know this.\n\nThe sum of % sometimes exceeds 100%, hence the author should check the rounding for each value in the graphs.\n\nWhen respondents were given the chance to answer multiple options, the choice of a Pie chart in % is questionable. Probably (see specific answers) those answers categories that aim to be discussed should be pulled out as well on their individual frequency of answer, for which a % distribution would make more sense.\n\nGeneral comments on: Results and Discussion:\nQ1.1: treating all words in the respondent's answers separately may denature the meaning of the answers.\nAlso, duplicates in the word cloud could dilute specific answers (capital letter issue?). If possible, it would be interesting to get another layer of interpretation to see for example how many general categories of respondents are represented, e.g. industry vs. public, management level vs. staff vs. PhD student & postdocs, etc...\n\nQ1.4 is not clearly analysed in my opinion.\nThe question aims at distributing the work time of respondents spent on activities identified in Q1.3, but the weighting factor could bias the total sum of time.\n\nThe options given to respondents to quantify their time were: all of their time is valued 100%(8), most: 75%(6), half: 50%(4), some: 12.5%(1), none: 0%(0)\n\nHowever, if a person answers (see e.g. n30 in Zenodo data): Image Analysis User: Some. Bioimage Analyst: Some. Then the total estimated time which is counted here is 25%. Problem: we don't know what that person does with the rest (75%) of the worktime, and the analysis of the data does not seem to count it. Hence the global impression (pie chart, figure) is misleading, suggesting for example that all respondents spend, on average, only 34.2% of their time on other tasks than Image analysis, which is wrong. Respondent n30 spends 75% of time on something else, but it doesn't seem to be counted. Each answer therefore sums a different amount of time, which makes the final % distribution of the figure somehow biased. I would like to see, therefore, how much absolute time of the respondents is spent on Image analysis, so as to support the conclusion of the paragraph. This could be done by estimating a new category \"rest of the time\", possibly to be fused with \"Other\" when respondents have quantified this category for their working time.\n\nAnother doubt, with the same origin of bias I believe:\nIn the answers, how can the 35.4% of subjects answering Q1.3 with \"Image Analysis Users\" convert into 2.3% of time spent on \"Using Image Analysis\"? Assume those 35.4% would spend minimally \"some time\", then the % should be at least 12.5% of 35.4% = 4.4%. Here, possibly, a crossed plot between Q1.3 and Q1.4 would be very informative to see how users spend their time on analysis, how many are intensive users (half of their time and above), etc... for each category.\n\nQ1.5: There are two questions in one.\nThe author should clarify how the distinction of \"who is interested in pursuing image analysis as potential career\" is made: is it by answering the question? Was the question optional? \n\nQ2.4:\nThis question reports very interesting results, redistributed against facility or research-oriented groups categories. However, given that the respondents could select more than one option, the distribution of the results in % is questionable. Here, to support the conclusions, the author could draw the absolute % against the total # of respondents for each specific answer: e.g. how many of the respondents have ticked \"Trainer\" respect to the total number of respondents in the specific category. I expect those individual % to increase for each response, hence reinforcing the conclusions. \n\nQ2.6: Probably, the acronyms should be expanded in the legend so as to ease the reading for non-specialist readers (e.g. probably out of UK).\nAlso, readers not aware of the different funders in the UK will probably have a hard time understanding the different nature of the funds (whether for research projects, infrastructure or other). Colors in the graph could orient the reader and provide a more detailed qualitative interpretation here. Although more difficult to draw, but maybe accessible in the current data, it could be interesting to get a sense for whether public vs. private funding contribute more or less to Bioimage Analysis.\n\nQ2.7: This question is very important and unique, it captures the stability of jobs in Bioimage Analysis. The answers to this question could, ideally, be discussed in the discussion part of the paper, to try to evaluate or compare to other communities/professional groups in Life Science and see if Bioimage Analysis related positions suffers a bias towards short term contracts.\n\nQ2.8: Here again, a % distribution for a multiple choice question yields a Pie chart that may not be accurate. Q2.10: It could be interesting to redistribute the answers on the facility vs. research groups category.\nThe extended data is not easy to \"read/go through\", here some extracts of the \"own answers\" could be interesting to read, if they provide interesting arguments/examples. In general, if feasible, it would be interesting to do this exercise on all questions, e.g. setting \"inset\" pie charts, in each figure where the answers where \"data was pooled across job descriptions\". Even if the data would not be discussed, it would release the interested reader (from one category or the other) from the duty to get into python scripting against the raw data.\n\nConclusions:\nAre there examples of grants/funds/fellowships dedicated to mixed research/support, in UK or beyond? It is a major topic for this paper and some context/background/example would probably help illustrating the current possibilities, or reinforcing the current lack of such funding (if there is no example to feature).\n\nReferences: are very few. Are there any equivalent study/survey reports in the field of e.g. imaging facilities or other types of facilities that could be cited and where, maybe, data is similar and can serve to elaborate more discussion ?\nSpecific comments:\nPlease refer to NEUBIAS with capital letters.\n\nRefs:\nThis reference has a version on Zenodo: Miura (20211).\n\nBetter to use www.neubias.org as a landing pag for NEUBIAS.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "82872", "date": "19 Apr 2021", "name": "Julia Fernandez-Rodriguez", "expertise": [ "Reviewer Expertise Head of an Imaging Facility", "background in Cell and Molecular Biology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper has done an excellent job first to highlight the need for Bioimage Analysts, and second show clearly one of the important problems of the field: the funded scheme. Without a clear support from the funders (universities and research bodies) it will be impossible to provide career possibilities for these professionals. Currently the research environment in the UK, but also in the rest of Europe, does not formally recognise Bioimage Analysts as a discrete entity, in terms of its funding opportunities, and career development.\nIt is also quite interesting that the respondents find a good model for funding the mixture of: their independent research interest and the financial means to support the local community with bioimage analysis. This also seems a good strategy to define a Bioimage Analysts. This founding model will allow them to have an identity in both the academic and technical settings.\nI do think the paper has to be indexed to show the problems about the recognition of the Bioimage Analysts and to provide long-term career possibilities.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "82873", "date": "22 Apr 2021", "name": "Martin L. Jones", "expertise": [ "Reviewer Expertise Bioimage analysis", "microscope development", "software development", "electron microscopy" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article presents the results of two surveys carried out across the UK's community of researchers and facility staff using image analysis as part of their work. The analyses of the responses are presented along with the anonymised raw data. The \"Extended Data\" provides free text supplied by respondents, providing useful additional comments.\n\nThe responses provide very useful insights into the current situation of the role of bioimage analysts in the UK and the analysis of the responses is thorough and well presented. Taken along with the results of the referenced NEUBIAS survey, this kind of information will hopefully raise the profile of bioimage analyst as a unique role, as well as seed discussions both in the bioimage analyst community and the broader research community, regarding the development and sustainability of careers in the area. It feels to me as though there are many parallels with the Research Software Engineering community, which may provide some useful roadmaps for the development of the bioimage analyst community. Community engagement and advocacy appear to have been key in the establishment of the RSE role and hopefully articles like this and the referenced NEUBIAS survey results will help bioimage analysts along a similar path!\n\nSome specific minor comments:\nIn Abstract: Methods: \"Respondents were collected\" should be \"Responses were collected\" or \"Respondents' answers were collected\".\n\nNEUBIAS should be capitalised throughout.\n\nCOST should be capitalised throughout.\n\nIn Methods, \"Royal Software Engineers\" should presumably be \"Research Software Engineers\"?\n\nIn Methods, \"In questionnaire 1 a Wordcloud (1)\" should be \"… a Wordcloud (Figure 1)\" and ideally hyperlinked as elsewhere.\n\nInconsistent capitalisation \"questionnaire 1 vs Questionnaire 2\" at a few places through the document.\n\nGiven the hopefully international readership, it might be worth stating somewhere you're using dd/mm/yyyy date format, since many countries use different formats.\n\nIn the Twitter response, does \"6 Tweets\" mean quote-tweets?\n\nResults and discussion:\nQ1.1 - the closing single quotes are incorrectly formatted (and in a few other places).\n\nQ1.2 - opening single quotes are incorrectly formatted.\n\nQ1.3 - \"either as a Bioimage Analyst, Specialist Image Analyst but a few used the term Software Engineers\" should be \"either as a Bioimage Analyst or Specialist Image Analyst, but a few used the term Software Engineers\".\n\nQ1.5 - \"…their obvious job description\" doesn't sound right - obvious to whom? Should this be \"stated job description\"?\n\nQ2.4 makes the important distinction between facility- and research-centric responses and the fact that bioimage analyst is highly and equally represented in both groups supports the conclusions drawn.\n\nQ2.5 - I'm not sure that a good reference exists, but \"This is fairly typical in the life sciences…\" should ideally be backed up with a reference, or it should be stated if this is anecdotal.\n\nQ2.6 - it might be useful to list the funders in descending order of number of mentions (and state that it is so ordered). Also expanding the abbreviations would be useful for non-UK based readers as most are national funders only.\n\nQ2.9 - typo \"There replies were broad\". It would perhaps be interesting to see the breakdown of this question according to facility/research status as well, one might suspect that facility staff are more likely able to explicitly support their community.\n\nQ2.10 - it'd be interesting to see this split according to research/facility as well. As the main identified \"track\" subdivision, it may be useful to have separate results for all questions in fact (where applicable), either in the main document or in the Extended Data, with reference in the main text where differences are appreciable. Most readers will naturally fall into one camp or the other, so being able to quickly make comparisons may be beneficial, whereas questions with more polarised answers may give an artificially watered down consensus when combined. It feels to me as though these surveys have identified this research vs. facility difference as a key and interesting area and so full exposition of the results would likely be useful.\n\nReference 1 should be updated with the new DOI: 10.5281/zenodo.4648077\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-276
https://f1000research.com/articles/9-970/v1	11 Aug 20	{ "type": "Research Article", "title": "Affective rating of audio and video clips using the EmojiGrid", "authors": [ "Alexander Toet", "Jan B. F. van Erp", "Jan B. F. van Erp" ], "abstract": "Background: In this study we measured the affective appraisal of sounds and video clips using a newly developed graphical self-report tool: the EmojiGrid. The EmojiGrid is a square grid, labeled with emoji that express different degrees of valence and arousal. Users rate the valence and arousal of a given stimulus by simply clicking on the grid. Methods: In Experiment I, observers (N=150, 74 males, mean age=25.2±3.5) used the EmojiGrid to rate their affective appraisal of 77 validated sound clips from nine different semantic categories, covering a large area of the affective space. In Experiment II, observers (N=60, 32 males, mean age=24.5±3.3) used the EmojiGrid to rate their affective appraisal of 50 validated film fragments varying in positive and negative affect (20 positive, 20 negative, 10 neutral). Results: The results of this study show that for both sound and video, the agreement between the mean ratings obtained with the EmojiGrid and those obtained with an alternative and validated affective rating tool in previous studies in the literature, is excellent for valence and good for arousal. Our results also show the typical universal U-shaped relation between mean valence and arousal that is commonly observed for affective sensory stimuli, both for sound and video. Conclusions: We conclude that the EmojiGrid can be used as an affective self-report tool for the assessment of sound and video-evoked emotions.", "keywords": [ "affective response", "audio clips", "video clips", "EmojiGrid", "valence", "arousal" ], "content": "Introduction\n\nIn daily human life, visual and auditory input from our environment significantly determines our feelings, behavior and evaluations (Fazio, 2001; Jaquet et al., 2014; Turley & Milliman, 2000, for a review see: Schreuder et al., 2016). The assessment of the affective response of users to the auditory and visual characteristics of for instance (built and natural) environments (Anderson et al., 1983; Huang et al., 2014; Kuijsters et al., 2015; Ma & Thompson, 2015; Medvedev et al., 2015; Toet et al., 2016; Watts & Pheasant, 2015) and their virtual representations (Houtkamp & Junger, 2010; Houtkamp et al., 2008; Rohrmann & Bishop, 2002; Toet et al., 2013; Westerdahl et al., 2006), multimedia content (Baveye et al., 2018; Soleymani et al., 2015), human-computer interaction systems (Fagerberg et al., 2004; Hudlicka, 2003; Jaimes & Sebe, 2010; Peter & Herbon, 2006; Pfister et al., 2011) and (serious) games (Anolli et al., 2010; Ekman & Lankoski, 2009; Garner et al., 2010; Geslin et al., 2016; Tsukamoto et al., 2010; Wolfson & Case, 2000) is an essential part of their design and evaluation and requires efficient methods to assess whether the desired experiences are indeed achieved. A wide range of physiological, behavioral and cognitive measures is currently available to measure the affective response to sensorial stimuli, each with their own advantages and disadvantages (for a review see: Kaneko et al., 2018a). The most practical and widely used instruments to measure affective responses are questionnaires and rating scales. However, their application is typically time-consuming and requires a significant amount of mental effort (people typically find it difficult to name their emotions, especially mixed or complex ones), which affects the experience itself (Constantinou et al., 2014; Lieberman, 2019; Lieberman et al., 2011; Taylor et al., 2003; Thomassin et al., 2012; for a review see: Torre & Lieberman, 2018) and restricts repeated application. While verbal rating scales are typically more efficient than questionnaires, they also require mental effort since users are required to relate their affective state to verbal descriptions (labels). Graphical rating tools however allow users to intuitively project their feelings to figural elements that correspond to their current affective state.\n\nArousal and pleasantness (valence) are principal dimensions of affective responses to environmental stimuli (Mehrabian & Russell, 1974). A popular graphical affective self-report tool is the Self-Assessment Mannikin (SAM) (Bradley & Lang, 1994): a set of iconic humanoid figures representing different degrees of valence, arousal, and dominance. Users respond by selecting from each of the three scales the figure that best expresses their own feeling. The SAM has previously been used for the affective rating of video fragments (e.g., Bos et al., 2013; Deng et al., 2017; Detenber et al., 2000; Detenber et al., 1998; Ellard et al., 2012; Ellis & Simons, 2005; Fernández et al., 2012; Soleymani et al., 2008) and auditory stimuli (e.g., Bergman et al., 2009; Bradley & Lang, 2000; Lemaitre et al., 2012; Morris & Boone, 1998; Redondo et al., 2008; Vastfjall et al., 2012). Although the SAM is validated and widely used, users often misunderstand the depicted emotions (Hayashi et al., 2016; Yusoff et al., 2013): especially the arousal dimension (shown as an ‘explosion’ in the belly area) is often interpreted incorrectly (Betella & Verschure, 2016; Broekens & Brinkman, 2013; Chen et al., 2018; Toet et al., 2018). The SAM also requires a successive assessment of the stimulus on each of its individual dimensions. To overcome these problems we developed an alternative intuitive graphical self-report tool to measure valence and arousal: the EmojiGrid (Toet et al., 2018). The EmojiGrid is a square grid (resembling the Affect Grid: Russell et al., 1989), labeled with emoji that express various degrees of valence and arousal. Users rate the valence and arousal of a given stimulus by simply clicking on the grid. It has been found that the use of emoji as scale anchors facilitates affective over cognitive responses (Phan et al., 2019). Previous studies on the assessment of affective responses to food images (Toet et al., 2018) and odorants (Toet et al., 2019) showed that the EmojiGrid is self-explaining: valence and arousal ratings did not depend on framing and verbal instructions (Kaneko et al., 2019; Toet et al., 2018). The current study was performed to investigate the EmojiGrid for the affective appraisal of auditory and visual stimuli.\n\nSounds can induce a wide range of affective and physiological responses (Bradley & Lang, 2000; Gomez & Danuser, 2004; Redondo et al., 2008). Ecological sounds have a clear association with objects or events. However, music can also elicit emotional responses that are as vivid and intense as emotions that are elicited by real-world events (Altenmüller et al., 2002; Gabrielsson & Lindström, 2003; Krumhansl, 1997) and can activate brain regions associated with reward, motivation, pleasure and the mediation of dopaminergic levels (Blood & Zatorre, 2001; Brown et al., 2004; Menon & Levitin, 2005; Small et al., 2001). Even abstract or highly simplified sounds can convey different emotions (Mion et al., 2010; Vastfjall et al., 2012) and can elicit vivid affective mental images when they have some salient acoustic properties in common with the actual sounds. As a result, auditory perception is emotionally biased (Tajadura-Jiménez et al., 2010; Tajadura-Jiménez & Västfjäll, 2008). Video clips can also effectively evoke various affective and physiological responses (Aguado et al., 2018; Carvalho et al., 2012; Rottenberg et al., 2007; Schaefer et al., 2010). While sounds and imagery individually elicit various affective responses that recruit similar brain structures (Gerdes et al., 2014), a wide range of non-linear interactions at multiple processing levels in the brain make that their combined effects are not a priori evident (e.g., Spreckelmeyer et al., 2006; for a review see: Schreuder et al., 2016). Several standardized and validated affective databases have been presented to enable a systematic investigation of sound (Bradley & Lang, 1999; Yang et al., 2018) and video (Aguado et al., 2018; Carvalho et al., 2012; Hewig et al., 2005; Schaefer et al., 2010) elicited affective responses.\n\nThis study evaluates the EmojiGrid as a self-report tool for the affective appraisal of auditory and visual events. In two experiments, participants were presented with different sound and video clips, covering both a large part of the valence scale and a wide range of semantic categories. The video clips were stripped of their sound channel (silent) to avoid interaction effects. After perceiving each stimulus, participants reported their affective appraisal (valence and arousal) using the EmojiGrid. The sound samples (Yang et al., 2018) and video clips (Aguado et al., 2018) had been validated in previous studies in the literature using 9-point SAM affective rating scales. This enables an evaluation of the EmojiGrid by directly comparing the mean affective ratings obtained with it to those that were obtained with the SAM.\n\nIn this study we also investigate how the mean valence and arousal ratings for the different stimuli are related. Although the relation between valence and arousal for affective stimuli varies between individuals and cultures (Kuppens et al., 2017), it typically shows a quadratic (U-shaped) form across persons (i.e., at the group level): stimuli that are on average rated either high or low on valence are typically also rated as more arousing than stimuli that are on average rated near neutral on valence (Kuppens et al., 2013; Mattek et al., 2017). For the valence and arousal ratings obtained with the EmojiGrid, we therefore also investigate to what extent a quadratic form describes their relation at the group level.\n\n\nMethods\n\nEnglish speaking participants from the UK were recruited via the Prolific database (https://www.prolific.co/). Exclusion criteria were age (outside the range of 18–35 years old) and hearing or (color) vision deficiencies. No further attempts were made to eliminate any sampling bias.\n\nWe estimated the sample size required for this study with the “ICC.Sample.Size” R-package, assuming an ICC of 0.70 (generally considered as ‘moderate’: Landis & Koch, 1977), and determined that sample sizes of 57 (Experiment 1) and 23 (Experiment 2) would yield a 95% confidence interval of sufficient precision (±0.07; Landis & Koch, 1977). Because the current experiment was run online and not in a well-controlled laboratory environment, we aimed to recruit about 2–3 times the minimum required number of participants.\n\nThis study was approved by the by TNO Ethics Committee (Application nr: 2019-012), and was conducted in accordance with the Helsinki Declaration of 1975, as revised in 2013 (World Medical Association, 2013). Participants electronically signed an informed consent by clicking “I agree to participate in this study”, affirming that they were at least 18 years old and voluntarily participated in the study. The participants received a small financial compensation for their participation.\n\nDemographics. The participants in this study reported their nationality, gender and age.\n\nValence and arousal: the EmojiGrid. The EmojiGrid is a square grid (similar to the Affect Grid: Russell et al., 1989), labeled with emoji that express various degrees of valence and arousal (Figure 1). Users rate their affective appraisal (i.e., the valence and arousal) of a given stimulus by pointing and clicking at the location on the grid that that best represents their impression. The EmojiGrid was originally developed and validated for the affective appraisal of food stimuli, since the SAM appeared to be frequently misunderstood in that context (Toet et al., 2018). It has since also been used and validated for the affective appraisal of odors (Toet et al., 2019).\n\nThe iconic facial expressions range from disliking (unpleasant) via neutral to liking (pleasant) along the horizontal (valence) axis, while their intensity increases along the vertical (arousal) axis. This figure has been reproduced with permission from Toet et al., 2018.\n\nParticipants took part in two anonymous online surveys, created with the Gorilla experiment builder (Anwyl-Irvine et al., 2019). After thanking the participants for their interest, the surveys first gave a general introduction to the experiment. The instructions asked the participants to perform the survey on a computer or tablet (but not on a device with a small screen such as a smartphone) and to activate the full-screen mode of their browser. This served to maximize the resolution of the questionnaire and to prevent distractions by other programs running in the background. In Experiment I (sounds) the participants were asked to turn off any potentially disturbing sound sources in their room. Then the participants were informed that they would be presented with a given number of different stimuli (sounds in Experiment I and video clips in Experiment II) during the experiment and they were asked to rate their affective appraisal of each stimulus. The instructions also mentioned that it was important to respond seriously, while there would be no correct or incorrect answers. Participants could electronically sign an informed consent. By clicking “ I agree to participate in this study ”, they confirmed that they were at least 18 years old and that their participation was voluntary. The survey then continued with an assessment of the demographic variables (nationality, gender, age).\n\nNext, the participants were familiarized with the EmojiGrid. First, it was explained how the tool could be used to rate valence and arousal for each stimulus. The instructions were: “To respond, first place the cursor inside the grid on a position that best represents how you feel about the stimulus, and then click the mouse button.” Note that the dimensions of valence and arousal were not mentioned here. Then the participants performed two practice trials. In Experiment I, these practice trials also allowed the repeated playing of the sound stimulus. This was done to allow the participants to adjust the sound level of their computer system. The actual experiment started immediately after the practice trials. The stimuli were presented in random order. The participants rated each stimulus by clicking at the appropriate location on the EmojiGrid. The next stimulus appeared immediately after clicking. There were no time restrictions. On average, each experiment lasted about 15 minutes.\n\nExperiment I: Sounds\n\nThis experiment served to validate the EmojiGrid as a rating tool for the affective appraisal of sound-evoked emotions. Thereto, participants rated valence and arousal for a selection of sounds from a validated sound database using the EmojiGrid. The results are compared with the corresponding SAM ratings provided for each sound in the database.\n\nStimuli. The sound stimuli used in this experiment are 77 sound clips from the expanded version of the validated International Affective Digitized Sounds database (IADS-E, available upon request; Yang et al., 2018). The sound clips were selected from 9 different semantic categories: scenarios (2), breaking sounds (8), daily routine sounds (8), electric sounds (8), people (8), sound effects (8), transport (8), animals (9), and music (10). For all sounds, Yang et al. (2018) provided normative ratings for valence and arousal, obtained with 9-point SAM scales and collected by at least 22 participants from a total pool of 207 young Japanese adults (103 males, 104 females, mean age 21.3 years, SD=2.4). The selection used in the current study was such that the mean affective (valence and arousal) ratings provided for stimuli in the same semantic category were maximally distributed over the two-dimensional affective space (ranging from very negative like a car horn, hurricane sounds or sounds of vomiting, via neutral like people walking up a stairs, to very positive music). As a result, the entire stimulus set is a representative cross-section of the IADS-E covering a large area of the affective space. All sound clips had a fixed duration of 6s. The exact composition of the stimulus set is provided in the Supplementary Material. Each participant rated all sound clips.\n\nParticipants. A total of 150 persons (74 males, 76 females) participated in this experiment. Their mean age was 25.2 (SD= 3.5) years.\n\nExperiment II: Video clips\n\nThis experiment served to validate the EmojiGrid as a self-report tool for the assessment of emotions evoke by (silent) video clips. Participants rated valence and arousal for a selection of video clips from a validated set of video fragments using the EmojiGrid. The results are compared with the corresponding SAM ratings for the video clips (Aguado et al., 2018).\n\nStimuli. The stimuli comprised of a set of 50 film fragments with different affective content (20 positive ones like a coral reef with swimming fishes and jumping dolphins, 10 neutral ones like a man walking in the street or an elevator going down, and 20 negative ones like someone being attacked or a car accident scene). All video clips had a fixed duration of 10 s and were stripped of their soundtracks (for detailed information about the video clips and their availability see Aguado et al., 2018). Aguado et al. (2018) obtained normative ratings for valence and arousal, collected by 38 young adults (19 males, 19 females, mean age 22.3 years, SD=2.2) using 9-point SAM scales. In the present study, each participant rated all video clips using the EmojiGrid.\n\nParticipants. A total of 60 persons (32 males, 28 females) participated in this experiment. Their mean age was 24.5 (SD= 3.3) years.\n\nAll statistical analyses were performed with IBM SPSS Statistics 26 (www.ibm.com) for Windows. The computation of the intraclass correlation coefficient (ICC) estimates with their associated 95% confidence intervals was based on a mean-rating (k = 3), consistency, 2-way mixed-effects model (Koo & Li, 2016; Shrout & Fleiss, 1979). ICC values less than 0.5 indicate poor reliability, values between 0.5 and 0.75 suggest moderate reliability, values between 0.75 and 0.9 represent good reliability, while values greater than 0.9 indicate excellent reliability (Koo & Li, 2016; Landis & Koch, 1977). For all other analyses a probability level of p < 0.05 was considered to be statistically significant.\n\nMATLAB 2020a was used to further investigate the data. The mean valence and arousal responses were computed across all participants and for each of the stimuli. MATLAB’s Curve Fitting Toolbox (version 3.5.7) was used to compute a least-squares fit of a quadratic function to the data points. Adjusted R-squared values were calculated to quantify the agreement between the data and the quadratic fits.\n\n\nResults\n\nFigure 2 shows the relation between the mean valence and arousal ratings for the 77 IADS-E sounds used as stimuli in the current study, measured both with the EmojiGrid (this study) and with a 9-point SAM scale by Yang et al. (2018). The curves in this figure represent least-squares quadratic fits to the data points. The adjusted R-squared values are 0.62 for results obtained with the EmojiGrid and 0.22 for the SAM results. Hence, both methods yield a relation between mean valence and arousal ratings that can indeed be described by a quadratic (U-shaped) relation at the nomothetic (group) level.\n\nBlue circles represent data obtained with the SAM (Yang et al., 2018), while red dots represent data obtained with the EmojiGrid (this study). The curves represent quadratic fits to the corresponding data points.\n\nThe linear (two-tailed) Pearson correlation coefficients between the valence and arousal ratings obtained with the EmojiGrid (present study) and with the SAM (Yang et al., 2018) were, respectively, 0.881 and 0.760 (p<0.001). To further quantify the agreement between both rating tools we computed intraclass correlation coefficients (ICC) with their 95% confidence intervals for the mean valence and arousal ratings between both studies. The ICC value for valence is 0.936 [0.899–0.959] while the ICC for arousal is 0.793 [0.674–0.868], indicating both studies show an excellent agreement for valence and a good agreement for arousal (even though the current study was performed via the internet and therefore did not provide the amount of control over many experimental factors as one would have in a lab experiment).\n\nFigure 3 shows the relation between the mean valence and arousal ratings for the 50 video clips tested, obtained with the EmojiGrid (this study) and with a nine-point SAM scale (Aguado et al., 2018). The curves in this figure represent quadratic fits to the data points. The adjusted R-squared values are respectively 0.68 and 0.78. Hence, both methods yield a relation between mean valence and arousal ratings that can be described by a quadratic (U-shaped) relation at the nomothetic (group) level.\n\nBlue circles represent data obtained with the SAM (Aguado et al., 2018) while red dots represent data obtained with the EmojiGrid (this study). The curves show quadratic fits to the corresponding data points.\n\nThe linear (two-tailed) Pearson correlation coefficients between the valence and arousal ratings obtained with the EmojiGrid (present study) and with the SAM (Aguado et al., 2018) were respectively 0.963 and 0.624 (p<0.001). To further quantify the agreement between both rating tools we computed intraclass correlation coefficients (ICC) with their 95% confidence intervals for the mean valence and arousal ratings between both studies. The ICC value for valence is 0.981 [0.967 – 0.989] while the ICC for arousal is 0.721 [0.509 – 0.842], indicating both studies show an excellent agreement for valence and a good agreement for arousal.\n\nRaw data from each experiment are available as Underlying data (Toet, 2020).\n\n\nConclusion\n\nIn this study we evaluated the recently developed EmojiGrid self-report tool for the affective rating of sounds and video. In two experiments, observers rated their affective appraisal of sound and video clips using the EmojiGrid. The results show a close correspondence between the mean ratings obtained with the EmojiGrid and those obtained with the validated SAM tool in previous validation studies in the literature: the agreement is excellent for valence and good for arousal, both for sound and video. Also, for both sound and video, the EmojiGrid yields the universal U-shaped (quadratic) relation between mean valence and arousal that is typically observed for affective sensory stimuli. We conclude that the EmojiGrid is an efficient affective self-report tool for the assessment of sound and video-evoked emotions.\n\nFuture applications of the EmojiGrid may involve the real-time evaluation of affective events or the provision of affective feedback. For instance, in studies on affective communication in human-computer interaction (e.g., Tajadura-Jiménez & Västfjäll, 2008), the EmojiGrid can be deployed as a continuous response tool by moving a mouse-controlled cursor over the grid while logging the cursor coordinates. Such an implementation may also afford the affective annotation of multimedia (Chen et al., 2007; Runge et al., 2016), and could be useful for personalized affective video retrieval or recommender systems (Hanjalic & Xu, 2005; Koelstra et al., 2012; Lopatovska & Arapakis, 2011; Xu et al., 2008), for real-time affective appraisal of entertainment (Fleureau et al., 2012) or to provide affective input to serious gaming applications (Anolli et al., 2010) and affective music generation (Kim & André, 2004). Sensiks (www.sensiks.com) has adopted a simplified version of the EmojiGrid in its Sensory Reality Pod to enable the user to select and tune multisensory (visual, auditory, tactile and olfactory) affective experiences.\n\n\nData availability\n\nOpen Science Framework: Affective rating of audio and video clips using the EmojiGrid. https://doi.org/10.17605/OSF.IO/GTZH4 (Toet, 2020).\n\nFile ‘Results_sound_video’ (XLSX) contains the EmojiGrid co-ordinates selected by each participant following each stimulus.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nThe authors thank Dr. Wanlu Yang (Hiroshima University, Higashi-Hiroshima, Japan) for providing the IADS-E sound database, and Dr. Luis Aguado (Universidad Complutense de Madrid, Spain) for providing the validated movie clips.\n\n\nReferences\n\nAguado L, Fernández-Cahill M, Román FJ, et al.: Evaluative and psychophysiological responses to short film clips of different emotional content. 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Reference Source" }	[ { "id": "69208", "date": "01 Sep 2020", "name": "Wei Ming Jonathan Phan", "expertise": [ "Reviewer Expertise Survey methodology", "rating formats", "Emotions", "and Emojis." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for the opportunity to review the manuscript: “Affective rating of audio and video clips using the EmojiGrid.” This paper is primarily focused on validating the extension of a scale format (EmojiGrid) to a broader range of stimuli (audio and video). Overall, the paper makes some useful methodological contributions such as (1) the potentially greater ease for respondents for rating their emotions; (2) capturing both arousal and valence simultaneously; and (3) the use of more familiar contemporary symbols (emojis) compared to the SAM (Bradley & Lang, 1994).1 I do have a few suggestions and concerns regarding the paper.\n\n1. Limitation of the EmojiGrid in measuring single discrete emotions.\n\nThe EmojiGrid is useful for respondents when selecting which area of the grid corresponds to their current felt emotion. However, emotions are not bipolar in nature and can often co-occur together, e.g., feeling bitter-sweet (Larsen et al., 2001; Larsen & McGraw, 2014)2,3. Thus, the current form of the EmojiGrid is limited to assessing stimuli that invoke single discrete emotions and may not be as suited for assessing real-time affective reactions (e.g., to entertainment or news). This limitation can potentially be highlighted in the discussion. Importantly, this limitation can be solved by future and different operationalizations of the grid structure when mixed emotions are the object of inquiry.\n\n2. Details regarding the stimuli selected.\nRelated to the first point, I note that the majority of the stimuli in both experiments (in particular experiment 1) seem to have a moderate amount of valence and arousal. Without knowing which stimuli were used, it is difficult to assess whether the emotion felt by the respondent was truly neutral or potential mix of emotions. To help the reader, please include two things potentially using tables in the supplementary material if needed. First, a greater description of which stimuli selected was expected to invoke which emotion in terms of both valence and arousal for both experiments. Second, please use a different numbering/labeling/coloring scheme that corresponds to the stimuli instead of dots for figures 1 and 2 when comparing the results from this study to previous work. Both are important because it allows the reader to visually assess the extent an expected emotion of stimuli (e.g., high arousal and positive valence) truly maps onto the mean scores and for the potential discrepancy between the two scale formats for the same stimuli to be obvious. This is important for replication but also because there is a greater dispersion when the SAM rating format is used.\n\n3. Comparing current data and alternate (future) research design.\n\nWhen comparing data from the current experiments to previous experiments the regression estimates are locally optimized based on the sample used to generate them. Thus, a caveat and clarification to potentially include are that the comparisons made are akin to that of two independent samples. Relatedly, an alternate design to consider would be doing a 4-block repeated measures design. Where participants rate the same stimuli using the two rating formats twice as: 1. A then A 2. B then B 3. A then B 4. B then A Blocks 3 and 4 would allow more direct comparisons between two different rating formats, especially given the greater dispersion in ratings observed when the SAM format is used.\n\n4. Free response clicks within the EmojiGrid\nI note that participants are free to click anywhere within the space of the EmojiGrid. I am curious as to variability/freedom that having no fixed anchor points generates. When participants respond do they more typically engage in: (1) subconsciously select a point close to one of the 25 potential points implied by the 5 X 5 grid of emojis, or (2) freely select a space with the grid, e.g., selecting point that corresponds to 2.30 arousal and 5.80 in valence? I ask this because the reliability of a scale is linked to the number of response points available (Preston & Colman, 2000; Schutz & Rucker, 1975)4,5. If respondents are truly giving their ratings as (2) then greater reliability would be a potential additional advantage of using the EmojiGrid. If it were (1) the design of the EmojiGrid could include finer lines (i.e., more grid lines) to help respondents more easily locate their emotions on the Grid.\n\nMinor points\nNationality information was collected from participants how was this information used? What was the distribution of nationalities for the participants?\n\nI appreciate the way the authors determined their sample sizes.\n\nI enjoyed reading your paper and hope you will find my comments helpful!\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "6433", "date": "06 Apr 2021", "name": "Alexander Toet", "role": "Author Response", "response": "Dear Dr Phan, Thank you for your helpful suggestions and constructive remarks, which helped us to improve the quality of our initial draft paper. In addition, we appreciate the fact that you spent your valuable time on this review. 1. Mixed emotions We thank the reviewer for raising this important issue. We now address this limitation in the Conclusion section as follows: “A limitation of the EmojiGrid is the fact that it is based on the circumplex model of affect which posits that positive and negative feelings are mutually exclusive (Russell, 1980). Hence, in its present form, and similar to other affective self-report tools like the SAM or VAS scales, the EmojiGrid only allows the measurement of a single emotion at a time. However, emotions are not strictly bipolar and two or more same or opposite valenced emotions can co-occur together (Larsen & McGraw, 2014; Larsen, McGraw, & Cacioppo, 2001). Mixed emotions consisting of opposite feelings can in principle be registered with the EmojiGrid by allowing participants to enter multiple responses. “ 2. Stimulus details Thank you for bringing this limitation to our attention. We agree that a labelling scheme (e.g. using the original stimuli identifier) makes a visual comparison between the experiments much easier. We therefore replaced the original graph with labelled graphs in the paper to allows the readers to visually assess and verify the expected emotions induced by the stimuli. We also added correlation plots for the mean valence and arousal ratings obtained both with the SAM and EmojiGrid to enable a direct comparison within each dimension. In addition, we now also provide a more detailed description of the selected stimuli in a new set of Excel notebooks that we uploaded to the Open Science Framework These notebooks include a brief description of the nature and content of all stimuli, their original affective classification, and their mean valence and arousal values (1) as provided by the authors of the (sound and video) databases and (2) as measured in this study. The notebooks also contain several graphs in which each of the stimuli is represented by the index number for easy identification. The graphs include plots showing (1) the relation between the mean valence measures obtained with the SAM and EmojiGrid, (2) the relation between the mean arousal measures obtained with the SAM and EmojiGrid, (3) the relation between the mean valence and arousal measures obtained with the SAM, and (4) the relation between the mean valence and arousal measures obtained with the EmojiGrid. 3. Comparing data Thank you for pointing out this limitation. We now address this issue in the Conclusion section as follows: “Another limitation of this study is the fact that the comparison of the SAM and EmojiGrid ratings were based on ratings from different populations (akin to a comparison of two independent samples). Hence, our current regression estimates are optimized based on the particular samples that were used. Future studies should investigate a design in which the same participants use both self-report tools to rate the same set of stimuli. “ 4. Free response clicks Thank you for raising this potentially important issue. We plotted the raw response data for visual inspection. The overall response pattern appears truly random and shows no regularities or evidence for attraction to any of the individual emojis lining the grid area or to any of the grid lines inside the grid area. Minor points Thank you for drawing our attention to this omission. All participants had the UK nationality. We now report this in the text. Nationality information was collected to check if the participants adhered to the recruitment restrictions as specified through Prolific. Thank you for your positive appraisal. Your comments were quite valuable, and definitely served to improve the quality of our paper." } ] }, { "id": "76598", "date": "11 Jan 2021", "name": "Linda K Kaye", "expertise": [ "Reviewer Expertise Psychology of emoji", "cyberpsychology", "online behaviour" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting study that seeks to validate the EmojiGrid for use with auditory and video stimuli. Thank you to the authors for providing the research resources on OSF as this is helpful when reviewing the research. Overall, the research has merits but would benefit from being more detailed especially in the introductory and discussion sections. I also have a methodological query but this may be rectified from additional clarity in the writing of this section.\nThe introduction could do with additional literature about the emotional affordances of emoji. That is, the research is presented as assuming that emoji are emotional stimuli but does not provide a review of the literature which can support this. Interestingly, recent evidence (Kaye et al., 2021) suggests that emoji may not be processed emotionally on an implicit level, so the authors should be careful about their assumptions in this regard. Relevant sources that may be useful:\nBai, Q., Dan, Q., Mu, Z., & Yang, M. (2019). A systematic review of emoji: Current research and future perspectives. Frontiers in Psychology, 10, e2221.\n\ndoi:10.3389/fpsyg.2019.022211 Derks, D., Fischer, A. H., & Bos, A. E. R. (2008). The role of emotion in computer-mediated communication: A review. Computers in Human Behavior, 24 (3), 766-7852 Kaye, L. K., Rodriguez Cuadrado, S., Malone, S. A., Wall, H. J., Gaunt, E., Mulvey, A. L., & Graham, C. (2021). How emotional are emoji?: Exploring the effect of emotional valence on the processing of emoji stimuli. Computers in Human Behavior, 116, 1066483 Novak, P. K., Smailović, J., Sluban, B., & Mozetič, I. (2015). Sentiment of emojis. PLoS ONE, 10 (12), e01442964\n\nWith regards to the data presented (e.g., Fig 2), it is not made explicitly clear how numerical values were determined based on the responses from the EmojiGrid. E.g., how are each of the emoji symbols based on their position on the axis determined numerically? From Fig 1, it looks like this ranges from 1 to 5 based on the number of emoji on each axis. However, looking in the methodology, the SAM scale is outlined as being a 9-item response scale so it isn’t clear how Fig 2 & 3 can present the data from these two scales on the same axis if the response scales are different.\n\nThe discussion could benefit from further elaboration. E.g., To what extent do the findings contribute theoretically to the literature? What are the limitations of the work?\n\nMinor\nIn the methodology, it is more typical to use the term “participants” rather than “persons”\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6434", "date": "06 Apr 2021", "name": "Alexander Toet", "role": "Author Response", "response": "Dear Dr Kaye, Thank you for your critical remarks and valuable suggestions which definitely helped us to improve our initial draft paper. Also, we appreciate the fact that you spent your valuable time on this review. 1. Literature about the emotional affordances of emoji Thank you for this suggestion. We agree that reviewing literature about the emotional affordances of emoji will be a valuable addition to the Introduction, helping the reader to better place the current findings in their context. We therefore added the following text to the Introduction: “Emoji are facial icons that can elicit the same range of neural (Gantiva, Sotaquirá, Araujo, & Cuervo, 2020) and emotional (Moore, Steiner, & Conlan, 2013) responses as real human faces. In contrast to photographs, emoji are not associated with overgeneralization (the misattribution of emotions and traits to neutral human faces that merely bear a subtle structural resemblance to emotional expressions: Said, Sebe, & Todorov, 2009), or racial, cultural and sexual biases. Although some facial emoji can be poly-interpretable (Miller et al., 2016; Tigwell & Flatla, 2016) it has been found that emoji with similar facial expressions are typically attributed similar meanings (Jaeger & Ares, 2017; Moore et al., 2013) that are also to a large extent language independent (Kralj Novak, Smailović, Sluban, & Mozetič, 2015). Emoji have a wide range of different applications, amongst others in psychological research (Bai, Dan, Mu, & Yang, 2019). Emoji based rating tools are increasingly becoming popular tools as self-report instruments (Kaye, Malone, & Wall, 2017) to measure for instance user and consumer experience (e.g. www.emojiscore.com). Since facial expressions can communicate a wide variety of both basic and complex emotions emoji-based self-report tools may also afford the measurement and expression of mixed (complex) emotions that are otherwise hard to verbalize (Elder, 2018). However, while facial images and emoji are processed in a largely equivalent manner, suggesting that some non-verbal aspects of emoji are processed automatically, further research is required to establish whether they are also emotionally appraised on an implicit level (Kaye et al., 2021).” 2. How numerical values were determined Thank you for pointing out this omission. We now include the following explanation of the scaling in the section on data analysis: “The response data (i.e., the horizontal or valence and vertical or arousal coordinates of the check marks on the EmojiGrid) were quantified as integers between 0 and 550 (the size of the square EmojiGrid in pixels), and then scaled between 1 and 9 for comparison with the results of Yang et al. (2018) obtained with a 9-point SAM scale (Experiment I), or between 0 and 8 for comparison with the results of Aguado et al. (2018), also obtained with a 9-point SAM scale (Experiment II).” 3. Contribution and limitations We now address some limitations of the present study (e.g. related to the measurement of mixed emotions and the study design itself) in the Discussion section (see also our reply to the comments of dr Pham). 4. Minor points We replaced “persons” by “participants” throughout the text." } ] } ]	1	https://f1000research.com/articles/9-970
https://f1000research.com/articles/10-273/v1	06 Apr 21	{ "type": "Review", "title": "Is butyrate a natural alternative to dexamethasone in the management of CoVID-19?", "authors": [ "Nithin K. K", "Prakash Patil", "Satheesh Kumar Bhandary", "Vikram Haridas", "Suchetha Kumari N", "Sarathkumar E", "Praveenkumar Shetty", "Nithin K. K", "Prakash Patil", "Satheesh Kumar Bhandary", "Vikram Haridas", "Suchetha Kumari N", "Sarathkumar E" ], "abstract": "Coronavirus disease 2019 (CoVID-19) caused by Severe Acute Respiratory Syndrome Coronavirus 2 has affected more than 100 million lives. Severe CoVID-19 infection may lead to acute respiratory distress syndrome and death of the patient, and is associated with hyperinflammation and cytokine storm. The broad spectrum immunosuppressant corticosteroid, dexamethasone, is being used to manage the cytokine storm and hyperinflammation in CoVID-19 patients. However, the extensive use of corticosteroids leads to serious adverse events and disruption of the gut-lung axis. Various micronutrients and probiotic supplementations are known to aid in the reduction of hyperinflammation and restoration of gut microbiota. The attenuation of the deleterious immune response and hyperinflammation could be mediated by short chain fatty acids produced by the gut microbiota. Butyric acid, the most extensively studied short chain fatty acid, is known for its anti-inflammatory properties. Additionally, butyric acid has been shown to ameliorate hyperinflammation and reduce oxidative stress in various pathologies, including respiratory viral infections. In this review, the potential anti-inflammatory effects of butyric acid that aid in cytokine storm depletion, and its usefulness in effective management of critical illness related to CoVID-19 have been discussed.", "keywords": [ "Butyrate", "Butyric acid", "CoVID-19", "Cytokine storm", "Dexamethasone", "Gut microbiota", "Hyperinflammation", "Probiotics" ], "content": "Introduction\n\nSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative factor for the Coronavirus disease 2019 (CoVID-19)1. SARS-CoV-2 has affected nearly 100 million people around the globe2. SARS-CoV-2 enters the host by binding to its receptor, angiotensin converting enzyme 2 (ACE2), which is expressed mainly in the lungs and intestine3–5. Upon infection, SARS-CoV-2 causes mild to severe inflammation, which disrupts homeostasis and the integrity of infected organs6,7. Furthermore, severe infection of CoVID-19 results in systemic inflammation, thrombosis, acute respiratory distress syndrome (ARDS) and multiple organ failure, which may lead to death8–10. The corticosteroid immunosuppressant, dexamethasone, which attenuates hyperinflammation and cytokine storm, is being used to treat seriously ill CoVID-19 patients and has been found to improve survival in hospitalised patients11,12. However, the prolonged usage of dexamethasone causes serious adverse effects and gut dysbiosis13,14. Besides, the hyperinflammation and thrombotic complications associated with CoVID-19 can also be alleviated by various nutrients including vitamins, polyunsaturated fatty acids, minerals, and even amino acids15–20. The growing number of studies indicate the potential role of nutritional supplement, probiotics, and gut microbiome in mitigating the inflammation and in preventing viral infections including respiratory viral infections21. Alterations of the gut microbiome has been observed during SARS-CoV-2 infection, which significantly reduces the abundance of beneficial microbiome and its metabolites, such as short chain fatty acids (SCFAs) including butyric acid22–24.\n\nButyric acid or butyrate can act primarily as an anti-inflammatory molecule and various studies have reported its role in mitigating hyperinflammation via several mechanisms25–27. For the past several years, our group has worked on the role of proinflammatory regulators in the pathogenesis of various inflammatory disorders and identified that the role of histone deacetylase (HDAC) inhibitor in activating anti-inflammatory molecules. Further, this leads to the simultaneous down regulation of proinflammatory membrane receptors, downstream signalling molecules and respective cytokines, resulting in inflammatory homeostasis. Our in vitro preliminary experiments using various cell lines have revealed that the molecular mechanism of butyrate in neutralising inflammatory devastation, induction of anti-inflammatory molecular expression and its translocation to the site of action, is almost similar to dexamethasone28. Consequently, we hypothesise if the SCFA, butyric acid, a HDAC inhibitor, which is synthesized by the gut microbiota, could have strong anti-inflammatory functions with anti-fibrotic properties. Therefore, this article reviews the anti-inflammatory properties of butyric acid or butyrate and its associated molecular pathways involved in controlling the cytokine storm and hyperinflammation associated with SARS-CoV-2 infection. Based on the various positive reports, we presume that butyric acid possesses potent anti-inflammatory activity, which suggests it as an alternative to dexamethasone for the preventive management of primary and secondary complications related to CoVID-19.\n\n\nCoronavirus disease 2019\n\nThe CoVID-19 pandemic is caused by SARS-Cov-2, which belongs to genera β-coronaviruses and is the seventh known coronavirus to infect humans4. Spike protein of SARS-CoV-2 binds to ACE2, a type I membrane protein29 expressed in the lung, heart, kidney, and intestine3,30,31. The majority of SARS-CoV-2 infected cases present with mild symptoms like dry cough, sore throat, fatigue and fever9,10,32. Less common symptoms such as myalgia, expectoration, pharyngalgia, dizziness, nausea, headache, haemoptysis, diarrhoea, abdominal pain, and vomiting have also been reported. Lymphocytopenia along with elevated expression of C-reactive proteins (CRP) and inflammatory cytokines are also common9,10,32,33. Infection can progress into severe disease with dyspnoea, grinding glass-like abnormalities and patchy consolidation areas in lungs observed upon imaging; viral pneumonia usually appears after 2–3 weeks of infection 9,10,32,33. However, some patients have developed organ failure, septic shock, myocarditis, acute cardiac injury, arrhythmia, pulmonary oedema, severe pneumonia, acute kidney injury, and ARDS. Inflammation, oxidative stress, and fibrosis associated with CoVID-19 is perhaps partially mediated by angiotensin II (AngII), a substrate for ACE2, which degrades it to anti-inflammatory angiotensin (1–7). The accumulation of AngII results in hyperinflammation induced by nucleotide-binding oligomerization domain (NOD) like receptors family pyrin domain-containing 3 (NLRP3) inflammasome and nuclear factor kappa B (NF-κB) activation34. Disrupted immune response in CoVID-19 is further characterized by decreased expression of human leukocyte antigen D related (HLA-DR) on CD(cluster of differentiation)14 monocytes, accompanied by decrease in number of CD4 and CD19 lymphocytes, and natural killer cells along with the continuous production of proinflammatory tumour necrosis factor (TNF)-α and interleukin (IL)-6 secreted by circulating monocytes, subsequently leading to cytokine storm and hyperinflammation7. Hypercytokinemia and hyperinflammation associated with CoVID-19 results in acute lung injury, ARDS and death of the patients8,35,36. Furthermore, the SARS-CoV-2 infection may lead to liver injury and its dysfunction, and dysbiosis in the gut, where high expression of ACE2 is observed. Myocardial damage by interaction of SARS-CoV-2 with ACE2 expressed in cardiac pericytes has also been observed37. In addition, the high complication of disseminated intravascular coagulation is known to be associated with the severe form of CoVID-1938.\n\nSARS-CoV2 infection significantly alters gut microbiota, increasing the number of opportunistic pathogens such as Clostridium hathewayi, Actinomyces viscosus, Bacteroides nordii, Streptococcus, Rothia, Erysipelatoclostridium and Veillonella along with significant reduction in beneficial bacteria such as Lachnospiraceae bacterium 5_1_63FAA, Eubacterium rectale, Ruminococcus obeum, Fusicatenibacter, Eubacterium hallii, Anaerostipes, Agathobacter, Roseburia, Dorea formicigenerans, Clostridium butyricum, Clostridium leptum and Faecalibacterium prausnitzii, which includes butyric acid producing bacteria (BPB). Abundance of BPB is negatively correlated with inflammatory and thrombosis markers including CRP, Procalcitonin and D-dimer. However, the plethora of opportunistic Coprobacillus species, Clostridium ramosum and C. hathewayi are positively associated with the CoVID-19 severity, but beneficial species Alistipes onderdonkii and Faecalibacterium prausnitzii show negative correlation. In addition, the probiotic bacteria, Lactobacillus and Bifidobacterium are also decreased in CoVID-19 patients22,24,39. The resulting gut dysbiosis may lead to aberrant inflammation and increased severity of CoVID-19 due to the disruption in the gut-lung axis34,39–41. The reduction in BPB may impact lung inflammation and subsequent injury associated with CoVID-1942,43.\n\n\nNutrients in mitigating the Covid-19 pathogenesis\n\nNutrition and nutrients play a vital role in enhancing immune response along with reduction of inflammation and oxidative stress44–46. Better nutritional status of CoVID-19 patients is associated with less adverse outcomes18,47–52. Vitamin D is involved in reducing respiratory infections, such as influenza, and a reduced plasma 25-hydroxyvitamin D (25(OH)D) concentration in SARS-CoV-2 patients has been observed53. Moreover, people with vitamin D deficiency are at higher risk of getting infected with SARS-CoV-254,55. Co-supplementation of vitamin D along with glutathione precursor L-cysteine significantly increases serum 25(OH)D levels and augments vitamin D regulatory gene expression, which in turn reduces the oxidative stress and inflammatory responses in CoVID-19 patients56. Vitamin D supplementation in SARS-CoV-2 infected patients attenuates the production of proinflammatory cytokines like Interferon (IFN)-γ, IL-6, IL-2 and TNF-α by inhibiting NF-κB and other pathways57–59. CoVID-19 associated inflammatory signalling pathways including NF-κB, Mitogen-Activated Protein Kinase (MAPK) and phosphatidylinositol 3-kinase/ protein kinase B (PI3K/AKT) and innate immune response pathways, such as Toll-like signalling and NOD-like signalling modulation and regulation can be mediated by the combination of curcumin, vitamin C, and glycyrrhizic acid60. Vitamin C has been known to improve the immune condition by enhancing differentiation and proliferation of B- and T-cells, but severe vitamin C deficiency is associated with pneumonia and respiratory tract infections61. Intravenous administration of vitamin C can significantly decrease IL-6 levels62,63. Glycyrrhizic acid and curcumin exhibits anti-viral, anti-inflammation, anti-cancer, and immune system benefits60. The combination of vitamin D/magnesium/vitamin B12 significantly reduced the subsequent need for oxygen therapy and/or intensive care support in older CoVID-19 patients57. Vitamin B12 is crucial in maintaining the healthy gut microbiome which plays a vital role in immune responses57. Fat soluble vitamin E acts as an antioxidant that scavenges Reactive Oxygen species (ROS) and inhibits devastating effects of hyperinflammation64. Moreover, the supplementation of vitamin E stimulates T cell function and confers protection against upper respiratory infections65.\n\nSelenium is one of the key micronutrients known to positively impact CoVID-19 patient recovery66,67. Selenium status regulates the expression of glutathione peroxidase 1 (GPX1), a cytosolic selenoenzyme known for its antioxidative properties. The antioxidant enzyme GPX1 mitigates the production of ROS and further leading to mutations in the viral genome68. In addition, attenuating ROS also helps in the inhibition of proinflammatory NF-κB activation and further nuclear translocation69. Severe endothelial injury and widespread pulmonary micro thromboses are accompanied with platelet activation and aggregation in patients with severe CoVID-19 manifestations. The synthetic Rupatadine (histamine1 receptor antagonist) and natural flavonoids with anti-inflammatory properties are known to inhibit the platelet activating factor70. Elderly individuals with deficiency of nutrients, such as vitamin C, vitamin D, calcium, folate, and zinc are prone to increase severity of SARS-CoV-2 infection71. Folic acid may inhibit furin protease and inactivates chymotrypsin-like protease (3CLpro)72. Zinc (Zn2+) deficiency contributes to impaired cell mediated immune response and increased susceptibility to various infections. However, increased intracellular levels of Zn2+ disrupt viral RNA replication including SARS-CoV-2, where Zn2+ inhibits RNA (Ribonucleic acid) dependent RNA polymerase (RdRp) elongation and template binding73. Among CoVID-19 patients, iron deficiency is strongly associated with increased inflammation and longer stay in hospitals74.\n\nHealth beneficial compounds, including minerals, antioxidants, phytochemicals, vitamins, and minerals present in fruits and vegetables, can exert antioxidative, anti-inflammatory and antiviral effects during various non-infectious and infectious disease71. Alliin, an S-allyl cysteine sulfoxide compound present in garlic has shown to have inhibitory action on 3CLpro, a protease that plays a vital role in SARS-CoV-2 replication75. Salvianolic acid A and curcumin have the potential to bind to 3CLpro with greater affinity76. Resveratrol acts as an anti-inflammatory molecule that inhibits the NFκB pathway and thereby reduces circulatory cytokines, such as IL-6 and TNF-α levels, which are observed in severe SARS-CoV-2 infection77. Sea cucumber (Stichopus japonicus) derived sulphated polysaccharide showed significant anti-viral activity against SARS-CoV-2 infection78. Omega-3 polyunsaturated fatty acids, including eicosapentaenoic acid and docosahexaenoic acid have been shown to exhibit anti-inflammatory effects by downregulation of the NF-κB pathway71,79,80. Free fatty acids such as oleic acid, arachidonic acid and linoleic acid have shown antiviral activity at micromolar concentrations81. Dietary fibre intake alters the intestinal microflora and enhances relative proportion of SCFAs, which exhibit anti-inflammatory properties through fatty acid receptors like G-protein-coupled receptor (GPCR) 41 and 4382–84.\n\n\nProbiotics: suppressors of respiratory tract infections and inflammation\n\nProbiotics are living microorganisms that provide health benefits to the host upon administration at appropriate doses85. Probiotics exert a wide range of beneficial effects such as host microbiome balancing, stimulation of immune system, enhancement of intestinal barrier function or inhibiting pathogens by direct interactions40,46,86,87 (Table 1). Several microorganisms belonging to the family of Enterococcus species (E. fecalis, E. faecium), Bifidobacterium species (B. bifidum, B. longum, B. lactis), Lactobacillus species (L. acidophilus, L. casei, L. rhamnosus), and Saccharomyces (S. boulardii, S. cerevisiae) are considered as probiotics40. Probiotic supplementation causes significant reduction in the incidence of oral and respiratory tract infections88,89. Dietary supplementation of cow’s milk and fermented rice with L. paracasei CBA L74 helps in prevention of common infectious disease including upper respiratory tract infections in children90. Daily intake of fermented milk containing probiotic L. casei strain ‘Shirota’ has been shown to reduce the incidence and duration of respiratory tract infections in healthy middle aged office workers and young children via modulation of the immune system91,92. Daily ingestion of the probiotic L. paracasei ST11 can reduce the degree of virus replication and dissemination thereby attenuating lung inflammation and subsequent death in mice infected with vaccinia virus95. L. gasseri SBT2055 exhibits antiviral activity against human respiratory syncytial virus (RSV) by silencing SWI2/SNF2-related cAMP Response Element-Binding Protein (CREB)-binding protein activator protein, which is involved in RSV replication. L. gasseri SBT2055 reduced the expression of proinflammatory cytokines in lungs upon RSV infection96. CC chemokine receptor 2 acts as a receptor for monocyte chemoattractant protein-1 (MCP-1), which induces increased lung inflammation and subsequently decreases survival associated with influenza virus infection. Prophylactic oral administration of heat-killed E. faecalis can protect mice from influenza virus infection and subsequent lung inflammation by modulation of MCP-1 production. Alternatively, lipoteichoic acid of E. faecalis binds to toll like receptor 2 and exerts antiviral and anti-inflammatory activity during influenza infection97. Oral administration of probiotics L. paracasei, L. gasseri, and B. longum improved immune response and reduced mortality in influenza infected mice105 by reducing the inflammation and oxidative stress associated with it106,107.\n\nProbiotics, in combination with enteral nutrition, given to post-operative gastric cancer patients aids in increased production of antibodies and reduction of inflammatory cytokines108. Oral administration of L. plantarum ameliorates intestinal inflammation and lipid metabolism disorders by modulating gut microbiota in turn producing more SCFAs in high-fat diet induced obese mice100. This disrupted enterohepatic immunoregulation, which can be ameliorated by intervention of Clostridium butyricum B1 via its metabolite butyric acid99. Probiotic mixture of Lactobacillus and Bifidobacterium prevents the non-alcoholic fatty liver disease by suppressing systemic adiposity and inflammation through butyric acid and its receptor GPR109A98. Treatment with probiotic strain L. acidophilus DDS-1 upsurges the abundance of beneficial bacteria such as Lactobacillus spp and Akkermansia spp and also the levels of butyrate, while downregulating the production of inflammatory cytokines IL-6, IL-1β, IL-1α, MCP-1, Macrophage Inflammatory Protein (MIP)-1α, MIP-1β, IL-12 and IFN-γ in aging mice101. L. paracasei KW3110 suppresses hyperinflammation via activation of M2 macrophages and exhibit anti-inflammatory effects via suppression of IL-β production and caspase 1 activation by promoting IL-10 production103. Probiotic complex of L. acidophilus, L. casei, L. fermentum, L. paracasei, Streptococcus thermophilus, Bifidobacterium longum, B. bifidum, B. breve, L. rhamnosus, L. plantarum, L. helveticus, and L. salivarius in combination with zinc and coenzyme Q10 can improve autoimmune arthritis via downregulation of proinflammatory cytokines including IL-6, IL-17 and TNF-α and inhibition of T-helper cell 17 (Th17) cell differentiation109–111. Oral administration of B. infantis suppresses allergic inflammation in lungs by significantly reducing serum levels of Immunoglobulin (Ig)E, IgG1, IL-4 and IL-13102. Daily administration of L. plantarum DR7 for 12 weeks can prevent development of upper respiratory tract infections among young adults through various mechanisms including inhibition of respiratory infection causing bacteria such as Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes and Streptococcus mutans, stimulation of proinflammatory cytokine production such as IL-10 and IL-4, and enhancement of antioxidant potential of RBC membrane93. Significant reduction in the number of Bifidobacteria and Lactobacilli along with increased number of Escherichia coli is observed in the gut of children with recurrent respiratory tract infections. Oral probiotic supplement containing Bifidobacterium infantis, L. acidophilus, E. faecalis and Bacillus cereus restored the intestinal flora along with reduction in incidence of respiratory tract infections and use of antibiotics23.\n\nRTIs-Respiratory tract infections; URTIs- Upper respiratory tract infections; CFU-Colony forming unit; RSV-Respiratory syncytial virus; CCR2- C-C chemokine receptor type 2; IL-Interleukin; IFN-Interferon; TNF-Tumour necrosis factor, SCFAs-short chain fatty acids; CoVID19- Coronavirus disease 2019. ↓-Reduce; ↑-Enhance; ↔- Balance.\n\nExopolysaccharides produced during milk fermentation by probiotic L. paracasei acts as a substrate for the gut microbiome. Fermentation of this exopolysaccharide increases the number of beneficial microbiomes belonging to phyla Firmicutes and Lentisphaerae, accompanied by the decrease in Actinobacteria, Proteobacteria and Bacteroidetes. Fermentation of exopolysaccharide enhances SCFAs production mainly butyric acid112. Aqueous probiotic supplements containing L. acidophilus NCIMB 30175, L. plantarum NCIMB 30173, L. rhamnosus NCIMB 30174 and E. faecium NCIMB 30176 induces an increase in butyric acid producing bacteria resulting in increased production of butyric acid exhibiting immunomodulatory activity via downregulation of proinflammatory cytokines such as MCP-1, Chemokine (C-X-C motif) ligand (CXCL)-10 and IL-8 in vitro113. Oral administration of multistrain probiotic mixture containing L. helveticus DSM 32242, B. lactis DSM 32246, L. paracasei DSM 32243, L. plantarum DSM 32244, L. brevis DSM 27961, L. acidophilus DSM 32241, Streptococcus thermophilus DSM 32345 and B. lactis DSM 32247 decreased development of respiratory failure associated with CoVID-19 by 8 times along with reduction in other symptoms such as diarrhoea, fever, asthenia, headache, myalgia, and dyspnoea104,114. Use of probiotics may restore the healthy gut microbiome in CoVID-19 patients and exhibit antiviral effects through gut-lung axis. The immunomodulatory role of probiotics helps in viral shedding, regulation of hypercytokinemia and associated multiple organ failure in severe CoVID-19 cases40,115–117.\n\n\nIs butyrate an alternative to dexamethasone?\n\nDexamethasone is a synthetic corticosteroid that acts as an anti-inflammatory agent, widely affecting innate and acquired immune system via glucocorticoid receptor118,119. Low dose dexamethasone treatment significantly supresses neutrophil infiltration and subsequent pulmonary inflammation and significantly improves lung function in early phase of ARDS120. Lower respiratory tract transcriptomic profiling of patients with CoVID-19 associated ARDS shows dysregulated immunoregulation and inflammation. This dysregulated immune response can be modulated by dexamethasone121. A short course of dexamethasone significantly reduces CRP levels and accelerates recovery122. Dexamethasone treatment in CoVID-19 patients who were receiving mechanical ventilation support results in lower mortality rate123. Severe CoVID-19 cases have been brought to remission state after 6 mg once a day intravenous administration of dexamethasone124.\n\nDexamethasone is indicated as a therapeutic option for immune thrombocytopenic purpura associated with CoVID-19124,125. Administration of dexamethasone before 30 hours of ARDS onset can significantly reduce the period of mechanical ventilation and mortality12. Dexamethasone provides an excellent protective effect against hypoxia associated with CoVID-19118. Intravenous dexamethasone treatment for CoVID-19 patients along with standard care significantly decreases the number of ventilator dependent days over 28 days11. High dose pulse therapy of dexamethasone increased the survival rate in CoVID-19 patients presented with hyperinflammation126. However, dexamethasone, a broad spectrum immunosuppressant, inhibits lymphocytes function and prevents macrophage mediated removal of apoptotic cells, which leads to reduced viral shedding and increases subsequent viremia in mild to moderately ill CoVID-19 patients124,127,128. Prolonged use of corticosteroids is associated with serious adverse effects such as short-term hyperglycaemia, cataracts, glaucoma, hypertension, psychological effects, weight gain, increased risk of secondary infections and osteoporosis13,129. Use of such corticosteroids may induce gut dysbiosis14.\n\nIntestinal microflora widely affects host health and alterations in the gut microbiome is correlated with several disease including respiratory disease130. Commensal gut microbiome and its metabolites can modulate host immunity and can also impact on pro inflammatory and immune-regulatory response131. Increased production of microbiome metabolite SCFAs may improve health condition132. Depletion of SCFA production makes mice more susceptible for allergic lung inflammation. Biological effects exerted by SCFAs is dependent mainly on two mechanisms: SCFA mediated (i) activation of GPCRs and (ii) inhibition of HDAC. SCFAs, via HDAC inhibition, positively impacts the functions and numbers of T-helper 1 cells, T-regulatory cells, and Th17 effector cells resulting in reduced inflammatory response in airway diseases130. The short chain fatty acid, butyrate or butyric acid is produced in the colon by anaerobic bacteria such as Roseburia intestinalis, Faecalibacterium prausnitzii, Clostridium butyricum, Megasphaera elsdenii, Mitsuokella multiacida, Eubacterium spp., Fusobacterium spp., Butyrivibrio spp. and Eubacterium hallii133. Butyrate concentration in the colon can reach from 10 to 20 mM and serves as major source of energy for colonocytes. Sodium butyrate supplementation enhances the abundance of beneficial bacteria such as Coprococcus, Lachnospiraceae, Ruminococcus, Bifidobacteriaceae and Actinobacteria improving intestinal barrier integrity in obese mice134.\n\nPrimarily, butyric acid exhibits anti-inflammatory and tissue protective function in the large intestine135. Butyric acid is a potential inhibitor of pro-inflammatory molecule NF-κB135–137 (Figure 1). Tight junction protein expression in intestinal epithelial cells is also influenced by butyrate mediated regulation138. Butyrate treatment on epithelial colon cells significantly downregulated the proinflammatory molecules including Toll-like receptor (TLR)2, TLR4, IL-6, IL-12A, IL-1β, IL-18, TNF, MAPK13, MAPK10, MAPK3, AKT1, AKT2, AKT3, NF-κB1A, NF-κB1, CXCL1, CXCL2, CXCL3, CXCL6, CXCL8, Chemokine ligands (CCL)2, Serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 1 (SERPINA1), SERPINA2, Colony Stimulating Factor (CSF) 3, Intercellular Adhesion Molecule 1 (ICAM1), Vascular Endothelial Growth Factor A (VEGFA), Major Vault Protein (MVP), Cathelicidin Antimicrobial Peptide (CAMP) and insulin-like growth factor binding protein (IGFBP)3, along with inhibition of proinflammatory pathways, including (i) triggering receptor expressed on myeloid cells (TREM-1) signalling, (ii) production of nitric oxide (NO) and ROS, (iii) high-mobility group box-1 (HMGB1) signalling, (iv) IL-6 signalling, and (v) acute phase response signalling25. Pre-treatment with butyric acid can attenuate heart depression along with reduction in inflammation and oxidative stress associated with septic shock in mice139. Acute lung injury along with ARDS characterized by excessive inflammation can be induced by various factors such as endotoxins, infections, hypoxia and complement activation. Lipopolysaccharide (LPS) induced acute lung injury (ALI) and inflammation can be attenuated by 4-phenyl butyric acid (4-PBA), a derivative of butyric acid and also by sodium butyrate26,140.\n\nThe downstream activators of these pathways induces the reactive oxygen species (ROS) generation and transcription factor, NF-κB dependent expression of proinflammatory molecules. HDACs, which deacetylates Signal transducer and activator of transcription 1 (STAT1), and promotes the nuclear translocation and subsequent activity of NF-κB. Target genes of NF-κB, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 increases the NF-κB activity via positive feedback loop. Histone deacetylase (HDAC) inhibitor, butyrate mediates its effects through GPCRs: Free fatty acid receptors 2/3 and GPCR 109A or by directly binding to HDAC active sites. Inhibition of NF-κB activity by butyrate attenuates inflammation and oxidative stress associated with various pathologies including CoVID-19. Butyrate also activates the transcription factor B lymphocyte-induced maturation protein-1 (BLIMP-1) and enhances the production of anti-inflammatory cytokines.\n\nProphylactic treatment of sodium butyrate significantly reduces myeloperoxidase activity and inflammatory cell infiltration into lungs which is correlated with the inhibition of proinflammatory cytokine, HMGB1 expression and NFκB26. The TLR 4/NF-κB pathway involved in the LPS is targeted by sodium butyrate, which attenuates the LPS induced lung injury27. Hyaluronan ester with butyric acid treatment induces apoptosis in mesangial cells after exposure to oxidative stress and thereby reducing cell proliferation via p38 MAPK pathway141. N-(1-carbamoyl-2-phenyl-ethyl) butyramide (FBA), a butyrate releasing compound, confers protection to mice from colitis induced by dextran sodium sulphate by suppressing neutrophils recruitment and subsequent release of pro-inflammatory molecules mediated by HDAC-9/NF-κB inhibition and peroxisome proliferator-activated receptor gamma (PPAR-γ) upregulation142. Butyrate inhibits IL-13 and IL-15 production by Type 2 innate lymphoid cells. Butyrate downregulates various RNA binding proteins and thereby post transcriptionally downregulating the expression of inflammatory genes143. Sodium butyrate attenuates AngII induced hypertension, cardiac hypertrophy, cardiac fibrosis, and inflammation by inhibiting Cyclooxygenase-2 (COX2)/ Prostaglandin E2 (PGE2) pathway in a HDAC5/ HDAC6 dependent manner144. Butyrate reduces AngII induced endothelial dysfunction145. Sodium butyrate attenuates lung inflammation by promoting forkhead box P3 (FOXP3) expression and suppression of IL-9 expression. Butyrate also reduces the infiltration of proinflammatory Th9 cells and eosinophils into lungs146. Mice treated with butyrate exhibited a significant reduction of inflammatory infiltrates in the airways, tissue, and vascular disruption, and subsequently less haemorrhaging in the lungs induced by influenza infection82. HDAC inhibitor sodium butyrate can suppress ACE2 expression in gut epithelial cells which can help in reducing gastrointestinal symptoms associated with CoVID-19147.\n\nPancreatitis and associated fibrosis induced by L-Arginine can be attenuated by sodium butyrate, which reduces collagen deposition and nitric oxide along with inhibition of profibrotic pancreatic stellate cells148. Butyric acid ameliorates bleomycin induced pulmonary fibrosis by attenuating leukocytes infiltration, oxidative stress and NF-κB activation149.\n\nConsequently, based on the evidence presented, the potential anti-inflammatory and tissue protective effects of butyric acid on lungs and gut, along with its ability to modulate gut microbiome diversity, enhancing production of endogenous butyric acid could be a better preventive approach to manage CoVID-19 over dexamethasone (Figure 2). However, there is a need for more detailed studies and clinical trials to determine the potency and long-term effect of butyric acid in the preventive management of seriously ill CoVID-19 patients.\n\nA. SARS-CoV-2 transmitted through aerosols reach the lungs via respiratory tract and enters the host cell by binding to its receptor, ACE2 present on the surface of pneumocytes. Followed by endosome mediated internalization, SARS-CoV-2 causes cell injury and subsequent hyperinflammation and cytokine storm, resulting in fibrosis of lungs. These cytokines reach the gut via blood and lymphatic vessels that instigates local inflammation in gut, ushering to leaky gut and gut dysbiosis, resulting in diarrhoea and malabsorption together with reduced production of short chain fatty acids. B. Dexamethasone a synthetic broad-spectrum immunosuppressant can inhibit cytokine storm associated with CoVID-19. As an alternative, oral administration of probiotics or gut microbiome metabolite, SCFAs may ameliorate gut inflammation, restore gut integrity, and gut microbiome. This enhances the production of endogenous SCFAs and reaches the lungs via blood and lymphatic vessels, and may inhibit hyperinflammation and cytokine storm along with induction of anti-inflammatory cytokines production which recovers the lung from injury and the acute respiratory distresses associated with CoVID-19.\n\n\nConclusion\n\nSeriously ill CoVID-19 patients are succumbing to respiratory distress syndrome due to significant hyperinflammation and cytokine storm. A broad-spectrum immunosuppressant, dexamethasone, is widely used to treat such cases. However, the prolonged use of this corticosteroid leads to severe adverse events and disrupted immune responses. There are growing number of advanced research studies in search of an alternative to dexamethasone for the better management of critical CoVID-19 patients. Hence, this review extensively searched for evidence to show the anti-inflammatory properties of butyric acid or butyrate and its associated molecular pathways involved in preventing SARS-CoV-2 infected patients from cytokine storm and hyperinflammation. It has been observed that the SARS-CoV-2 infection significantly decreases butyric acid producing bacteria in the host gut. Further, previous research shows that a histone deacetylase inhibitor, butyric acid has proven to be anti-inflammatory in lung inflammation including inflammation associated with respiratory viral infection. Therefore, based on the various positive reports, we presume that butyric acid possesses potent anti-inflammatory activity, making it a suitable alternative candidate for the preventive management of primary and secondary complications related to CoVID-19.\n\n\nData availabilty\n\nNo data is associated with this article.", "appendix": "References\n\nZu ZY, Di Jiang M, Xu PP, et al.: Coronavirus Disease 2019 (COVID-19): A Perspective from China. Radiology. 2020; 296(2): E15–E25. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUniversity JH: COVID-19 Case Tracker. Johns Hopkins University; 2019; [cited 2020 12 April]. This website is a resource to help advance the understanding of the virus, inform the public, and brief policymakers in order to guide a response, improve care, and save lives.]. Reference Source\n\nHoffmann M, Kleine-Weber H, Schroeder S, et al.: SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nd'Ettorre G, Ceccarelli G, Marazzato M, et al.: Challenges in the Management of SARS-CoV2 Infection: The Role of Oral Bacteriotherapy as Complementary Therapeutic Strategy to Avoid the Progression of COVID-19. Front Med (Lausanne). 2020; 7: 389. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLehtoranta L, Latvala S, Lehtinen MJ: Role of Probiotics in Stimulating the Immune System in Viral Respiratory Tract Infections: A Narrative Review. Nutrients. 2020; 12(10): 3163. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVaghef-Mehrabany E, Homayouni-Rad A, Alipour B, et al.: Effects of Probiotic Supplementation on Oxidative Stress Indices in Women with Rheumatoid Arthritis: A Randomized Double-Blind Clinical Trial. J Am Coll Nutr. 2016; 35(4): 291–9. PubMed Abstract \| Publisher Full Text\n\nTamtaji OR, Kouchaki E, Salami M, et al.: The Effects of Probiotic Supplementation on Gene Expression Related to Inflammation, Insulin, and Lipids in Patients With Multiple Sclerosis: A Randomized, Double-Blind, Placebo-Controlled Trial. J Am Coll Nutr. 2017; 36(8): 660–665. PubMed Abstract \| Publisher Full Text\n\nXie H, Lu Q, Wang H, et al.: Effects of probiotics combined with enteral nutrition on immune function and inflammatory response in postoperative patients with gastric cancer. J BUON. 2018; 23(3): 678–83. PubMed Abstract\n\nLee SY, Lee SH, Jhun J, et al.: A Combination with Probiotic Complex, Zinc, and Coenzyme Q10 Attenuates Autoimmune Arthritis by Regulation of Th17/Treg Balance. J Med Food. 2018; 21(1): 39–46. PubMed Abstract \| Publisher Full Text\n\nJia H, Ren S, Wang X: Heat-killed probiotic regulates the body's regulatory immunity to attenuate subsequent experimental autoimmune arthritis. Immunol Lett. 2019; 216: 89–96. PubMed Abstract \| Publisher Full Text\n\nVaghef-Mehrabany E, Alipour B, Homayouni-Rad A, et al.: Probiotic supplementation improves inflammatory status in patients with rheumatoid arthritis. Nutrition. 2014; 30(4): 430–5. PubMed Abstract \| Publisher Full Text\n\nBengoa AA, Dardis C, Gagliarini N, et al.: Exopolysaccharides From Lactobacillus paracasei Isolated From Kefir as Potential Bioactive Compounds for Microbiota Modulation. Front Microbiol. 2020; 11: 583254. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoens F, Van den Abbeele P, Basit AW, et al.: A four-strain probiotic exerts positive immunomodulatory effects by enhancing colonic butyrate production in vitro. Int J Pharm. 2019; 555: 1–10. PubMed Abstract \| Publisher Full Text\n\nWalton GE, Gibson GR, Hunter KA: Mechanisms linking the human gut microbiome to prophylactic and treatment strategies for COVID-19. Br J Nutr. 2020; 1–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMahooti M, Miri SM, Abdolalipour E, et al.: The immunomodulatory effects of probiotics on respiratory viral infections: A hint for COVID-19 treatment? Microb Pathog. 2020; 148: 104452. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGohil K, Samson R, Dastager S, et al.: Probiotics in the prophylaxis of COVID-19: something is better than nothing. 3 Biotech. 2021; 11(1): 1. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBaindara P, Chakraborty R, Holliday ZM, et al.: Oral probiotics in coronavirus disease 2019: connecting the gut-lung axis to viral pathogenesis, inflammation, secondary infection and clinical trials. New Microbes New Infect. 2021; 40: 100837. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHosseinzadeh MH, Shamshirian A, Ebrahimzadeh MA: Dexamethasone vs COVID-19: An experimental study in line with the preliminary findings of a large trial. Int J Clin Pract. 2020; e13943. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAndreakos E, Papadaki M, Serhan CN: Dexamethasone, pro-resolving lipid mediators and resolution of inflammation in COVID-19. Allergy. 2021; 76(3): 626–628. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMikolka P, Kosutova P, Kolomaznik M, et al.: Effect of different dosages of dexamethasone therapy on lung function and inflammation in an early phase of acute respiratory distress syndrome model. Physiol Res. 2019; 68(Suppl 3): S253–S63. PubMed Abstract \| Publisher Full Text\n\nSarma A, Christenson S, Mick E, et al.: COVID-19 ARDS is characterized by a dysregulated host response that differs from cytokine storm and is modified by dexamethasone. Res Sq. 2021; rs.3.rs–141578. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSelvaraj V, Dapaah-Afriyie K, Finn A, et al.: Short-Term Dexamethasone in Sars-CoV-2 Patients. R I Med J (2013). 2020; 103(6): 39–43. PubMed Abstract\n\nRECOVERY Collaborative Group; Horby P, Lim WS, et al.: Dexamethasone in Hospitalized Patients with Covid-19. N Engl J Med. 2021; 384(8): 693–704. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHassan ME, Hasan HM, Sridharan K, et al.: Dexamethasone in severe COVID-19 infection: A case series. Respir Med Case Rep. 2020; 31: 101205. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLévesque V, Millaire É, Corsilli D, et al.: Severe immune thrombocytopenic purpura in critical COVID-19. Int J Hematol. 2020; 112(5): 746–50. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZúñiga MÁL, Moreno-Moral A, Ocaña-Granados A, et al.: High-dose corticosteroid pulse therapy increases the survival rate in COVID-19 patients at risk of hyper-inflammatory response. PLoS One. 2021; 16(1): e0243964. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWaterer GW, Rello J: Steroids and COVID-19: We Need a Precision Approach, Not One Size Fits All. Infect Dis Ther. 2020; 9(4): 701–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSharun K, Tiwari R, Dhama J, et al.: Dexamethasone to combat cytokine storm in COVID-19: Clinical trials and preliminary evidence. Int J Surg. 2020; 82: 179–81. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSalem MA: A Response to the Recommendations for Using Dexamethasone for the Treatment of COVID-19: The Dark Side of Dexamethasone. J Pharm Pract. 2021; 34(2): 179–180. PubMed Abstract \| Publisher Full Text\n\nHe LH, Ren LF, Li JF, et al.: Intestinal Flora as a Potential Strategy to Fight SARS-CoV-2 Infection. Front Microbiol. 2020; 11: 1388. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhou X, Du L, Shi R, et al.: Early-life food nutrition, microbiota maturation and immune development shape life-long health. Crit Rev Food Sci Nutr. 2019; 59(sup1): S30–S8. PubMed Abstract \| Publisher Full Text\n\nTayyeb JZ, Popeijus HE, Mensink RP, et al.: Butyric Acid Added Apically to Intestinal Caco-2 Cells Elevates Hepatic ApoA-I Transcription and Rescues Lower ApoA-I Expression in Inflamed HepG2 Cells Co-Cultured in the Basolateral Compartment. Biomolecules. 2021; 11(1): 71. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPituch A, Walkowiak J, Banaszkiewicz A: Butyric acid in functional constipation. Prz Gastroenterol. 2013; 8(5): 295–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFang W, Xue H, Chen X, et al.: Supplementation with Sodium Butyrate Modulates the Composition of the Gut Microbiota and Ameliorates High-Fat Diet-Induced Obesity in Mice. J Nutr. 2019; 149(5): 747–754. PubMed Abstract \| Publisher Full Text\n\nLiu J, Zhu H, Li B, et al.: Beneficial effects of butyrate in intestinal injury. J Pediatr Surg. 2020; 55(6): 1088–1093. PubMed Abstract \| Publisher Full Text\n\nBachmann M, Meissner C, Pfeilschifter J, et al.: Cooperation between the bacterial-derived short-chain fatty acid butyrate and interleukin-22 detected in human Caco2 colon epithelial/carcinoma cells. Biofactors. 2017; 43(2): 283–292. PubMed Abstract \| Publisher Full Text\n\nJohnstone M, Bennett N, Standifer C, et al.: Characterization of the Pro-Inflammatory Cytokine IL-1β on Butyrate Oxidation in Colorectal Cancer Cells. J Cell Biochem. 2017; 118(6): 1614–1621. PubMed Abstract \| Publisher Full Text\n\nYin J, Zhou C, Yang K, et al.: Mutual regulation between butyrate and hypoxia-inducible factor-1α in epithelial cell promotes expression of tight junction proteins. Cell Biol Int. 2020; 44(6): 1405–1414. PubMed Abstract \| Publisher Full Text\n\nWang F, Jin Z, Shen K, et al.: Butyrate pretreatment attenuates heart depression in a mice model of endotoxin-induced sepsis via anti-inflammation and anti-oxidation. Am J Emerg Med. 2017; 35(3): 402–409. PubMed Abstract \| Publisher Full Text\n\nZeng M, Sang W, Chen S, et al.: 4-PBA inhibits LPS-induced inflammation through regulating ER stress and autophagy in acute lung injury models. Toxicol Lett. 2017; 271: 26–37. PubMed Abstract \| Publisher Full Text\n\nBaraldi O, Bianchi F, Menghi V, et al.: An in vitro model of renal inflammation after ischemic oxidative stress injury: nephroprotective effects of a hyaluronan ester with butyric acid on mesangial cells. J Inflamm Res. 2017; 10: 135–142. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSimeoli R, Raso GM, Pirozzi C, et al.: An orally administered butyrate-releasing derivative reduces neutrophil recruitment and inflammation in dextran sulphate sodium-induced murine colitis. Br J Pharmacol. 2017; 174(11): 1484–1496. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTorun A, Enayat S, Sheraj I, et al.: Butyrate mediated regulation of RNA binding proteins in the post-transcriptional regulation of inflammatory gene expression. Cell Signal. 2019; 64: 109410. PubMed Abstract \| Publisher Full Text\n\nZhang L, Deng M, Lu A, et al.: Sodium butyrate attenuates angiotensin II-induced cardiac hypertrophy by inhibiting COX2/PGE2 pathway via a HDAC5/HDAC6-dependent mechanism. J Cell Mol Med. 2019; 23(12): 8139–8150. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRobles-Vera I, Toral M, de la Visitacion N, et al.: Protective Effects of Short-Chain Fatty Acids on Endothelial Dysfunction Induced by Angiotensin II. Front Physiol. 2020; 11: 277. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nde Souza Vieira R, Castoldi A, Basso PJ, et al.: Butyrate Attenuates Lung Inflammation by Negatively Modulating Th9 Cells. Front Immunol. 2019; 10: 67. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTakahashi Y, Hayakawa A, Sano R, et al.: Histone deacetylase inhibitors suppress ACE2 and ABO simultaneously, suggesting a preventive potential against COVID-19. Sci Rep. 2021; 11(1): 3379. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKanika G, Khan S, Jena G: Sodium Butyrate Ameliorates L-Arginine-Induced Pancreatitis and Associated Fibrosis in Wistar Rat: Role of Inflammation and Nitrosative Stress. J Biochem Mol Toxicol. 2015; 29(8): 349–59. PubMed Abstract \| Publisher Full Text\n\nKabel AM, Omar MS, Elmaaboud MAA: Amelioration of bleomycin-induced lung fibrosis in rats by valproic acid and butyrate: Role of nuclear factor kappa-B, proinflammatory cytokines and oxidative stress. Int Immunopharmacol. 2016; 39: 335–342. PubMed Abstract \| Publisher Full Text" }	[ { "id": "82836", "date": "06 May 2021", "name": "Mallika Valapala", "expertise": [ "Reviewer Expertise Cell biology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe review article by Nithin et al. discusses whether butyrate can be a natural alternative to dexamethasone in the management of COVID-19. The manuscript is well written and with a clear explanation of purpose. The authors illustrate divergent signaling pathways associated with the pathophysiology of COVID-19. They also point out the issues that dexamethasone has and why butyrate can be an alternative. Listed below are some of my concerns:\nThe authors mention many benefits of butyrate in this review. I suggest the authors should also mention whether there are any side effects of butyrate in specific groups, such as pregnant women and those with underlying conditions. Such a description would enable a more comprehensive assessment of this compound.\n\nThe authors provided a schematic of pro-inflammatory intracellular signaling pathways and in same schematic describe the mechanism of action of butyrate. There are many signaling pathways in this schematic and readers are easy to get confused. I suggest the authors simplify the schematic or separate it into two different schematics, one for butyrate regulation and another one for the regulation of pro-inflammatory mediators.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? No\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [] }, { "id": "82767", "date": "07 May 2021", "name": "Stephen O. Mathew", "expertise": [ "Reviewer Expertise Tumor immunology", "NK cell biology", "innate immunity" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors have written a comprehensive review on the role of butyrate as a natural alternative to dexamethasone in the management of COVID-19. Various micronutrients and probiotic supplementations are known to aid in the reduction of hyperinflammation and restoration of gut microbiota. The attenuation of the deleterious immune response and hyperinflammation could be mediated by short chain fatty acids (SCFA) produced by the gut microbiota. Butyric acid is well known for its anti-inflammatory properties and ability to reduce oxidative stress and hyperinflammation in many diseases. In this review, the potential anti-inflammatory effects of butyric acid that aid in cytokine storm depletion, and its usefulness in effective management of critical illness related to COVID-19 have been discussed.\nWhile the review is well written it seems like the title does not fully reflect the content of the review. More than 50% of the review talks about gut microbiome and probiotics and nutrients that play a role in COVID-19 pathogenesis which is not reflected in the title. Only in the second half they talk about the role of butyrate. The title needs to be revised to fully reflect the article.\nThere are several new publications that discuss the role of butyrate in COVID-19 pathogenesis which could be added to make this a comprehensive review like J Li et al. (2021)1; Sarkar, P et al. (2020)2; Wang L et al. (2017)3.\nCOVID-19 pathogenesis is exacerbated in individuals who have co-morbidities. What is the role of butyrate in co-morbidities? This has not been discussed.\nInnate immune cells play a major role in the COVID-19 pathogenesis. Although the role of butyrate is described in adaptive immune response, its role in innate immunity in the context of COVID-19 pathogenesis is minimally mentioned.\nSome of the molecules like PGE2, ROS-IKK pathway has not been described in Figure 1 legend.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [] }, { "id": "82768", "date": "17 May 2021", "name": "Harishkumar Madhyastha", "expertise": [ "Reviewer Expertise Signal transduction" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAbstract can be rewritten, some sentences are incomplete. Various pathologies but only respiratory infection is mentioned.\nAuthors mentioned the usefulness of probiotics in management of tissue inflammation, some more clear idea on mechanism to mitigate the inflammation, particularly the viral load is demanded.\nChemistry of butyrate and dexamethasone and its structure activity relationship in preventing the cytokine storm is necessary.\nFigure 1 - over expressions of anti-inflammatory cytokines is mediated with STAT1 junction proteins and nuclear translocation. Is it possible mention the linking pathway between these?\nFigure 2 - whats is the linker molecules in Gut-Lung axis?\nFuture prospective at end would certainly increase the quality of the manuscript.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? No\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-273
https://f1000research.com/articles/9-1240/v1	15 Oct 20	{ "type": "Research Article", "title": "In vitro evaluation of the anti-diabetic potential of Helichrysum petiolare Hilliard & B.L. Burtt using HepG2 (C3A) and L6 cell lines", "authors": [ "Adebowale Emmanuel Aladejana", "Graeme Bradley", "Anthony Jide Afolayan", "Adebowale Emmanuel Aladejana", "Graeme Bradley" ], "abstract": "Background: Helichrysum petiolare Hilliard & B.L. Burtt has been listed in a survey of plants used in traditional medicine for the treatment of type 2 diabetes in the Eastern Cape of South Africa. In this study, the antidiabetic potentials of ethanol, cold aqueous (CAQ) and boiled aqueous (BAQ) extracts of H. petiolare were investigated. Methods: The cytotoxic and glucose utilization effects of the extracts were evaluated using L6 myocytes and HepG2 (C3A) hepatocytes. α-amylase, α-glucosidase and lipase inhibition assays were also carried out. Results: The ethanol extract showed significant cytotoxic effects in the treated cells. Both BAQ and CAQ extracts significantly increased glucose uptake in L6 and C3A cell lines. The CAQ extract enhanced glucose uptake more in the L6 myocytes than in the C3A cell-lines hepatocytes. The BAQ extract showed higher levels of inhibition on α–amylase and α-glucosidase than CAQ. The activities were not significantly different from acarbose. However, BAQ showed lower lipase inhibition than acarbose (p<0.05). Conclusions: The BAQ and CAQ extracts of H. petiolare may, therefore, contain pharmacologically active and relatively non-toxic hypoglycaemic chemicals, which may be effective substitutes in the treatment of diabetes mellitus.", "keywords": [ "Helichrysum petiolare Hilliard & B.L. Burtt", "myocytes", "α–amylase", "α-glucosidase", "hepatocytes" ], "content": "Introduction\n\nDiabetes mellitus (DM) is a complex multi-factorial metabolic anomaly characterized by glucose intolerance and hyperglycaemia, either due to relatively impaired insulin secretion or reduced effectiveness of insulin in facilitating glucose uptake1. DM can be categorized based on its aetiology and clinical symptoms into type 1 (insulin-dependent) diabetes mellitus (IDDM), and type 2 (non-insulin-dependent) diabetes mellitus (NIDDM). Type 2 diabetes mellitus is the foremost predominant form of diabetes and it is characterized by hyperglycaemia and abnormal blood lipid levels. The leading cause of this disease has been reported to be either congenital or unhealthy lifestyle or both2.\n\nThe worldwide prevalence of diabetes has increased drastically in the last 30 years, and the current statistics suggest the number is to double in the next 20 years3. According to the International Diabetes Federation (IDF), the disease influences well over 366 million (M) individuals around the world which, by 2030, is projected to increase to 552 M4. Nigeria with 3.2 M cases, South Africa with 2 M, Kenya with over 0.7 M, and Cameroon with over 0.5 M have been estimated to have the most elevated predominance of the disease in each sub-region of Africa4.\n\nThe liver and the skeletal muscle are some of the major tissues responsible for the maintenance of the body’s glucose homeostasis. Glucose gains access into the hepatocytes and the myocytes through GLUT2 and GLUT4 receptors, respectively. The GLUT2 receptor is a transmembrane carrier protein that enables protein facilitated glucose transport into the hepatocytes; it does not need insulin. The GLUT4 receptor, however, needs to be translocated through insulin activation from the endoplasmic reticulum to the plasma membrane to facilitate glucose uptake. A postprandial spike in blood glucose is accompanied by increased insulin secretion by the pancreas. The liver, under the influence of insulin, takes up postprandial glucose and stores it as glycogen through glycogenesis. High level of blood insulin, therefore, favours increased liver uptake of blood glucose for glycogenesis. A defect in hepatic glucose uptake and GLUT4 activity may result in insulin resistance, ultimately leading to the development of diabetes. Therefore, enhanced muscle and liver glucose uptake are considered primary therapeutic targets for diabetes management5.\n\nPrevention of diabetes through healthy lifestyles (e.g. healthy diet, increased activity, weight reduction, etc.) is better than a cure. To date, diabetes has no cure, and the common oral hypoglycaemic synthetic drugs currently used in its management are accompanied by inalienable side effects6. Researchers are now in search of alternative and complementary drugs to proffer enduring solutions to this metabolic disease, with limited side effects4. Many herbal remedies are effective anti-hyperglycemic agents and they are also a very important component of the health care conveyance framework in most African nations7. In one of their submissions, the World Health Organization (WHO) supported the assessment of medicinal plants (MPs) based on their viability, low cost and possession of very few adverse effects8. Plants are used for therapeutic purposes by almost 80% of South Africans; this is because of the non-affordability of orthodox medicine and rising costs conventional treatments4. However, not enough studies have been done to comprehensively evaluate the dosage, safety and anti-hyperglycemic potentials of most of the medicinal plants. There is, therefore, need to scientifically evaluate their toxicities and antidiabetic potentials through in vivo and/or in vitro studies before exposure to human subjects.\n\nSo far, surveys have reported over 44 plants in the Eastern Cape of South Africa used in traditional medicine to treat type 2 diabetes9,10. One of these herbal plants is Helichrysum petiolare Hilliard & B.L. Burtt, a plant commonly found in the Eastern Cape of South Africa, and traditionally known as imphepho9,10. A previous study has revealed H. petiolare as useful in herbal medicine for the treatment of diabetes, headache, reproductive problems, heart problems, respiratory infections, fever, high blood pressure, pain, and wounds11. According to the study, extracts and compounds obtained from H. petiolare possess antigenotoxic, antityrosinase, antioxidant, anti-inflammatory, and cytotoxic activities11. To date, very little is known of the safety and mechanism of action of H. petiolare. This study, therefore, evaluated the toxicity and anti-diabetic potentials of several whole plant extracts of H. petiolare using in vitro methods, intending to shed light on its mechanism of action.\n\n\nMethods\n\nThe whole plant of Helichrysum petiolare was procured from a Rastafarian who collected it from Hogsback, in Raymond Mhlaba Municipality of Eastern Cape. The purchased plant was identified and authenticated by Professor C.N. Cupido of the Department of Botany, University of Fort Hare, Alice, and indexed at the Giffen Herbarium, University of Fort Hare, Alice Campus, Eastern Cape, South Africa.\n\nThe whole plant of H. petiolare was washed, cleaned and oven-dried at 40°C. The dried sample was pulverized using an electrical blender and sieved (20 μm mesh). A portion (200 g) of the sample was then soaked individually in 1 L of ethanol and water (for cold aqueous (CAQ) extracts) and shaken on an orbital shaker for 24 hours, while another portion was boiled in 1 L of water (for boiled aqueous (BAQ) extract) for 15 minutes. The solution was then filtered using the Buchner funnel and Whatman No. 1 filter paper and concentrated at 78°C (for ethanol extract) using a Rotary vacuum evaporator (Scietek, Model: RE 300), while the concentration of the aqueous extracts was done using a freeze drier (Vir Tis benchtop K, Vir Tis Co., Gardiner, NY, USA). The concentrated extracts were stored at 4°C in the refrigerator until required12.\n\nHepG2 (C3A) hepatocytes (catalogue number: HB-8065) and L6 myocytes (catalogue number: JCRB9081) were purchased from Cellonex, South Africa. Fetal calf serum (FCS), Roswell Park Memorial Institute (RPMI) 1640 Medium, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and Dulbecco's Modified Eagle Medium (DMEM) were procured from GE Healthcare Life Sciences (Logan, UT, USA). Dimethyl sulfoxide (DMSO) and phosphate-buffered saline (PBS) (with and without Ca2+ and Mg2+ and trypsin) were purchased from Lonza (Walkersville, MD, USA). Bovine serum albumin (BSA), and insulin were purchased from Sigma (St. Louis, MO, USA).\n\nIncubation of all cell cultures in a humidified environment with 5% CO2 was performed at 37°C. Growth medium that consisted of RPMI 1640 medium and 10% fetal calf serum was added to the HepG2 cells every 2-3 days, and the L6 myoblast cells were cultivated in an antibiotic-free media containing RPMI 1640 and 10% fetal calf serum. The cell lines were subcultured post 90% confluence.\n\nTo determine cell viability, 100 µl MTT (0.5 mg/ml in DMEM) was added to each well and incubated at 37°C for an hour. The MTT solution was removed, and 200 µl DMSO was added. The mixture was again incubated for 20 minutes at room temperature, and the absorbance was read at 540 nm. The cytotoxicity was expressed as a percentage of untreated control13.\n\nUsing the method described by Van de Venter et al. (2008), L6 myocytes and HepG2 (C3A) hepatocytes were each seeded at a density of 5000 cells/well into separate 96-well plates14; a row was kept devoid of cells in each plate as the “no cell control.” The L6 cell medium was later replaced with differentiation medium (DMEM containing 2% horse serum) after approximately 80% confluence and then incubated for an additional five days to allow full differentiation. The C3A cells were also incubated for an additional 2-3 days to become confluent. The spent medium of both plates was removed and substituted with fresh medium containing the extract at 200 µl/well. Treatments were continued for 48 hours, after which the spent culture medium was aspirated and the cells were rinsed once with PBS. 50 µl of 8 mM glucose solution (RPMI medium diluted with PBS and supplemented with BSA to a final concentration of 0.1%) were added, while 1000 ng insulin and 1000 ng of metformin were also added for the L6 and C3A positive controls, respectively. The plates were returned to the incubator for 2 hours (L6 cells) or 3 hours (C3A cells), after which 5 µl was transferred from them to new plates and 200 µl glucose assay reagent (glucose oxidase/peroxidase colourimetric reagent) was added. The mixtures were again incubated at 37°C for 10–20 minutes and the absorbances were measured at 510 nm using a BioTek SynergyMx UV Spectrophotometer. Estimations of glucose utilisation were done as the differences between the test samples and the no cell controls and expressed as percentages of the untreated controls. Cell viability was determined in each well using the MTT assay13.\n\nThe α-amylase assay was carried out using the 3,5-dinitrosalicylic acid (DNSA) method described by Wickramaratne et al. (2016)15. The plant extract was dissolved in at least 10% DMSO and dissolution was further achieved in a buffer (pH 6.9, Na2HPO4 / NaH2PO4 [0.02 M], NaCl [0.006 M]) at 10 to 1000 μg/ml concentrations. A 200 μl volume of α-amylase (2 units/ml) was mixed with 200 μl extract and incubated at 30°C for 10 minutes. Thereafter, 200 μl of the starch solution (1% in water (w/v)) was added to each tube and incubated for 3 minutes. 200 μl DNSA reagent (12 g of sodium potassium tartrate tetrahydrate in 8.0 mL of 2 M NaOH and 20 mL of 96 mM of 3,5-dinitrosalicylic acid solution) was applied to terminate the reaction and the mixture was steamed in an 85–90°C water bath for 10 minutes. The blend was cooled down to optimum temperature and mixed with 5 ml of distilled water, and a UV Spectrophotometer (BioTek SynergyMx) was used to measure the absorbance at 540 nm. The blank with 100% enzyme activity was prepared with 200 μL of the buffer in place of the plant extract. Similarly, prepared was a sample blank, using the plant extract at each concentration in the absence of the enzyme solution. A positive control sample was prepared using acarbose (100 μg/ml–2 μg/ml) and the reaction was performed similarly to the reaction with plant extract as mentioned above. The inhibitory activity of α-amylase was expressed as a % inhibition and measured based on the below equation. The % inhibition of α-amylase was plotted against the concentrations of the extract and IC50 values were taken from the graph.\n\nThe procedure described by Sagbo et al. (2018) was used for the α-glucosidase inhibition assay. Summarily, 5 μl of the plant extract (prepared at a concentration of 50 µg/ml, 100 µg/ml, and 200 µg/ml) was applied to 20 μl solution of 50 μg / ml α-glucosidase in a 96-well plate. A 60 μl potassium phosphate buffer (pH 6.8) of 67 mM was added and the plate was further incubated for 5 minutes. 10 μl of 10 mM ρ-nitro-phenyl-α-D-glucoside (PNPGLUC), was added after incubation and incubated again at 37°C for 20 minutes. 25 µl of 100 mM Na2CO3 (sodium carbonate) solution was applied after incubation and the absorption measured at 405 nm using a UV-visible spectrophotometer (BioTek SynergyMx). In addition, a blank and sample blank are prepared by adding 5 μl of deionized water instead of plant extract and 20 μl of deionized water instead of the enzyme. Acarbose was used as a positive control and the percentage inhibition was determined by the following equation:\n\nUsing the method described by Jaradat et al. (2017), 0.1 ml of porcine pancreatic lipase (1 mg/ml, prepared immediately before use) was added to test tubes containing 0.2 ml of the various concentrations of plant extract (1 mg/ml stock solution in 10% DMSO, diluted to 30, 60, 120, 240, and 480 µg/ml)16. The resulting mixtures were then topped up to 1 ml with Tri-HCl solution (pH 7.4) and incubated for 15 minutes at 25°C. 0.1 ml of PNPB solution (20.9 mg of PNPB in 2 ml of acetonitrile) was added to each test tube after incubation, and the mixture was incubated again at 37°C for 30 minutes. Determination of pancreatic lipase activity was made by measuring the hydrolysis of p-nitro phenylbutyrate to p-nitrophenol at 405 nm using a UV-visible spectrophotometer (BioTek SynergyMx). The same procedure was repeated for the aqueous and organic extracts and for acarbose (a positive control) using the same concentrations as mentioned above. The tests were done in triplicates.\n\nThe obtained data were analysed for statistical significance using minitab-17 software. All experiments were repeated at least three times using different transfer numbers of C3A and L6 cells. The two-tailed Student t-test for two samples assuming equal variance was used to determine the statistical significance of each treatment. Error bars indicate SD of individual experiments, performed in quadruplicate.\n\n\nResults\n\nThe results of glucose utilization assays using the L6 muscle cells are shown in Figure 1. According to the results17, the CAQ extract, compared to the untreated and insulin-treated controls, induced a significant (p < 0.05) dose-dependent increase in glucose uptake across all the concentrations (Figure 1a). The effect of the BAQ extract on glucose uptake was not dose-dependent; it failed to induce any significant change at concentrations beyond 50 μg/ml (Figure 1c). The ethanol extract had a similar effect on the L6 cells’ glucose uptake as the CAQ, except at 100 μg/ml, where the rate of uptake plummets by roughly 50% (Figure 1e).\n\n(a) Effect of the CAQ extract of H. petiolare on glucose uptake in L6 myocytes (data are expressed as mean ± SD, n = 3); (b) MTT cytotoxicity of the plant’s CAQ extract on L6 myocytes (data are expressed as % of control ± SD, n = 3); (c) effect of the BAQ extract of H. petiolare on glucose uptake in L6 myocytes (data are expressed as mean ± SD, n = 3); (d) MTT cytotoxicity of the plant’s BAQ extract on L6 myocytes (data are expressed as % of control ± SD, n = 3); (e) effect of the ethanol extract of H. petiolare on glucose uptake in L6 myocytes (data are expressed as mean ± SD, n = 3); (f) MTT cytotoxicity of the plant’s ethanol extract on L6 myocytes (data are expressed as % of control ± SD, n = 3). * Indicates a significant difference (p < 0.05) compared to the untreated control, and # indicates no significant difference relative to the positive control (insulin).BAQ, boiled aqueous; CAQ, cold aqueous; ETQ, ethanol; Ins, insulin.\n\nThe MTT assay showed the CAQ and BAQ extracts, when compared to the untreated and insulin-treated controls, had no significant toxic effect on the viability of the L6 cells (Figures 1b and 1d). The ethanol extract, however, showed significant toxicity at 100 μg/ml, reducing the viability of the L6 cells by more than half.\n\nFigure 2 shows the results of glucose utilization assays using HepG2 (C3A) hepatocytes17. The CAQ treated cells showed significant dose-dependent increases in glucose uptake up to 50 μg/ml; the rate, however, dropped steeply by 26% at 100 μg/ml (Figure 2a). The result also showed concentration-dependent increases in glucose uptake in the BAQ treated cells (Figure 2c). These increases were significant (p < 0.05) at 50 μg/ml when compared to the metformin-treated control and also significant at 100 μg/ml when compared to the untreated and metformin-treated controls; the rate of glucose utilization appeared to slow down with the increase in concentration. No significant increase in glucose uptake was observed in the ethanol-treated cells across all concentrations (Figure 2e); in fact, the rate of glucose utilization of the cells dropped significantly at 100 μg/ml when compared to the untreated control.\n\n(a) Effect of the CAQ extract of H. petiolare on glucose uptake in HepG2 (C3A) hepatocytes (data are expressed as mean ± SD, n = 3); (b) MTT cytotoxicity of the plant’s CAQ extract on HepG2 cells (data are expressed as % of control ± SD, n = 3); (c) effect of the BAQ extract of H. petiolare on glucose uptake in HepG2 (C3A) hepatocytes (data are expressed as mean ± SD, n = 3); (d) MTT cytotoxicity of the plant’s BAQ extract on HepG2 cells (data are expressed as % of control ± SD, n = 3); (e) effect of the ethanol extract of H. petiolare on glucose uptake in HepG2 (C3A) hepatocytes (data are expressed as mean ± SD, n = 3); (f) MTT cytotoxicity of the plant’s ethanol extract on HepG2 cells (data are expressed as % of control ± SD, n = 3). * Indicates a significant difference (p < 0.05) compared to the untreated control, and # indicates no significant difference relative to the positive control (metformin). BAQ, boiled aqueous; CAQ, cold aqueous; ETQ, ethanol; Met, metformin.\n\nThe in vitro MTT cytotoxicity assay showed the CAQ and BAQ extracts of H. petiolare had no toxic effect on the HepG2 (C3A) cell lines across all concentrations compared to the untreated and metformin-treated controls (Figures 2b and 2d). The ethanol extract, however, showed significant (p < 0.05) toxicity at 100 μg/ml, where it reduced the cells’ viability by roughly 40% (Figure 2f).\n\nFigure 3 shows the inhibition of α-glucosidase activity using the BAQ and CAQ extracts, while Table 1 shows the IC50 values of the extracts17. According to the results, both extracts exhibited concentration-dependent inhibition of α-glucosidase activity compared to the standard acarbose (IC50 = 804.01 ± 27.09 µg/ml).\n\nData expressed as mean ± SD (n = 3). Columns with the same alphabet within the same concentration are not significantly different.\n\nValues within the same column followed by different superscript are significantly different (p < 0.05).\n\nThe inhibitory effects of the BAQ extract (IC50 = 844.27 ± 36.81 µg/ml) was, however, significantly (p < 0.05) higher than those of the CAQ extract (IC50 = 1075.39 ± 8.46 µg/ml), and significantly closer to those of the standard acarbose across all concentrations.\n\nThe results of the α-amylase inhibition assay (Figure 4 and Table 1) showed BAQ and CAQ extracts had concentration-dependent inhibitory effects on α-amylase activity across all concentrations17. The BAQ extract (IC50 = 0.361 ± 0.021 µg/ml) had the highest inhibitory effects, even higher than those of the standard acarbose (IC50 = 0.378 ± 0.0084 µg/ml) at 0.05 µg/ml and 0.4 µg/ml, respectively. The CAQ (IC50 = 0.383 ± 0.025 µg/ml) extract, however, was highest in its inhibitory effect at 0.1 µg/ml and 0.2 µg/ml. The inhibitory effects of both extracts appeared to flatten out from 0.4 µg/ml to 0.8 µg/ml, depicting that there was no apparent increase in the extracts’ inhibitory effects on α-amylase activity at concentrations greater than 0.4 µg/ml.\n\nData expressed as mean ± SD (n = 3). Columns with the same alphabet within the same concentration are not significantly different.\n\nThe results of the lipase inhibition assay using H. petiolare BAQ and CAQ extracts, as shown in Figure 5 and Table 1, showed significant concentration-dependent lipase inhibition in both extracts17. The BAQ extract (IC50 = 117.56 + 1.66 µg/ml) showed significantly (p < 0.05) higher lipase inhibition when compared with CAQ and the acarbose control; its inhibitory effect rose steadily from being the least at 30 μg/ml (BAQ<CAQ<acarbose), to the highest (BAQ>acarbose>CAQ) at 480 μg/ml. The CAQ (IC50 = 228.61 + 12.73 µg/ml) extract, however, displayed the least lipase inhibition across all the concentrations.\n\nData expressed as mean ± SD (n = 3). Columns with the same alphabet within the same concentration are not significantly different.\n\n\nDiscussion\n\nType 2 diabetes mellitus is a metabolic disease that is characterized by insulin resistance and hyperglycaemia. Long-term complications of the disease include macrovascular diseases (e.g. coronary artery disease, and cerebrovascular disease), microvascular diseases (e.g. diabetic retinopathy, and nephropathy), cataracts, erectile dysfunction, infections, diabetic ulcers, etc.2. The disease may be controlled and its complications prevented by bringing down the blood glucose level. This may be achieved through reduction of carbohydrate digestion/absorption and gluconeogenesis, and enhancement of glycogenesis and glucose uptake with minimal side effects and cytotoxicity.\n\nH. petiolare, as shown in this study, could be the effective anti-hyperglycemic agent much needed in achieving this aim. According to our results (Figure 1f), the ethanol extract of H. petiolare showed cytotoxic properties at high concentrations, reducing the viability of the myocytes by more than half at 100 μg/ml. Our previous work showed similar findings, in which the ethanol extract of H. petiolare was not only found to be mitotoxic and cytotoxic against C3A hepatocytes, but it was also found to have the potential to induce steatosis and phospholipidosis in the cells18. This further supports the findings of Lourens and co-workers, in their work where the chloroform:methanol (1:1) leaf and stem extracts of H. petiolare were found to be toxic against transformed human kidney epithelial (Graham) cells, MCF-7 breast adenocarcinoma, and SF-268 glioblastoma cells19. The cytotoxicity of the ethanol extract reflected its possible myotoxicity in human subjects, and also gave further explanation for the reduced glucose uptake (-50%) observed in the ethanol-treated L6 myocytes at 100 μg/ml (Figure 1e). The reduced glucose uptake might, however, be the result of a reduced cell population. The CAQ and BAQ extracts showed no cytotoxic effect on the L6 myocytes (Figure 1b and 1d). The results showed significantly enhanced glucose uptake in the CAQ and BAQ treated L6 cells (Figure 1a and 1c), most importantly for CAQ. The CAQ extract showed a similar hypoglycaemic effect on the myocytes to the hormone insulin (Figure 6). Insulin binds to the α-subunit of the tautomeric insulin receptor of the myocyte’s cell membrane, triggering a cascade of reactions which activates the translocation of the GLUT 4 receptor into the membrane to facilitate glucose transport into the cytoplasm2. Phytochemical compounds such as phenols, flavonoids, and flavanols have been reported to facilitate glucose uptake in cells through activation of insulin receptors20. Therefore, the enhanced glucose uptake induced by the extract may be as a result of phytochemicals (i.e. flavonoids, proanthocyanidins, flavonols, saponin, and alkaloids) previously reported by Aladejana et al. (2020) to be significantly high in H. petiolare21. The CAQ extract may, therefore, be effective in activating insulin receptors, enhancing glucose uptake in the myocytes, and making more glucose available for energy metabolism in exercising muscles; hence, reducing hyperglycaemia and its possible complications, ultimately halting type 2 diabetes.\n\nThe figure shows the levels of glucose uptake after treatment with H. petiolare. “↓” indicates decrease.\n\nCytotoxicity of the ethanol extracts on HepG2 (C3A) hepatocytes as shown in the MTT cytotoxicity assay (Figure 2f) was also similar to the findings of our previous study18, and suggests the potential hepato- and myotoxicity of the organic extracts of H. petiolare in human subjects. The toxicity of the ethanol extract and the consequent reduction in cell viability was possibly responsible for the 61% decrease in glucose utilization observed at 100 μg/ml in the ethanol extract-treated cells (Figure 2e). The lack of cytotoxicity of the CAQ and BAQ treated HepG2 (C3A) hepatocytes (Figure 2b and 2d) is reflected in the positive increases in glucose utilization in the cells (Figure 2a and 2c). However, despite no apparent sign of cytotoxicity, the degree of glucose utilization in the CAQ treated hepatocytes declined by 26% at 100 μg/ml; this may be due to negative feedback as a result of the high concentration of extract. The non-cytotoxicity and significant increases in glucose uptake observed in the BAQ and CAQ treated cells as compared to the metformin-treated control and the untreated control suggests the hypoglycaemic potentials of the extracts, most importantly, the BAQ extract (Figure 2). The BAQ extract induced hepatic glucose uptake like metformin (Figure 6). Metformin enhances hepatic glucose uptake by activation of the AMP-activated protein kinase (AMPK), this triggers a series of reactions which may lead to increased liver sensitivity to insulin1. H. petiolare is rich in phytochemicals which have been shown by Hanhineva and co-workers to increase hepatic glucose uptake and suppress the hepatic release of glucose22. The BAQ extract may, therefore, be effective in enhancing hepatic glucose uptake in human subjects, triggering glycogenesis or lipogenesis, preventing the hepatic release of glucose, and hence aiding the facilitation of glucose homeostasis.\n\nCarbohydrate absorption depends on the presence of amylase, disaccharidases, and normal intestinal mucosal cells with normal active transport mechanisms. The α-amylase enzyme is a glycoside hydrolase that catalyses the hydrolysis of starch at its α-1,4-glycosidic bonds into disaccharides and trisaccharides, which are further converted by other enzymes (e.g. disaccharidases) to glucose2. Alpha-glucosidase (e.g. maltase and sucrose) is a disaccharidase that acts upon α (1→4) bonds. It is found in the brush border of the small intestine where it acts on starch and disaccharides to hydrolyze terminal non-reducing α (1→4)-linked glucose residues to release a single α-glucose molecule (Figure 7)2. Glucose can be absorbed by the brush-border cells only if all three mechanisms are functioning. Any impairment to one of them may result in reduced or non-absorption of glucose from the intestine, and reduced levels of circulating blood glucose. The ethanol extract of H. petiolare was excluded from the enzyme inhibition assay due to its cytotoxicity. Inhibition of α–amylase and α-glucosidase activities by BAQ and CAQ extracts, as shown in the results (Figure 3 and Figure 4), suggests the extracts (most importantly the BAQ extract) may be effective in modulating postprandial hyperglycaemia by reducing the degree of glucose absorption from the small intestine by enterocytes.\n\nThe figure shows the hydrolysis of carbohydrate and triglyceride after treatment with H. petiolare respectively. “↓” indicates decrease, “o” represents a glucose molecule, and “*” represents a fructose molecule.\n\nBlood glucose levels are raised in the fasted state through gluconeogenesis, using non-glucose precursors e.g. amino acids, lactate, glycerol etc. Glycerol is derived from the hydrolysis of triglycerides by lipase enzymes. Pancreatic lipase hydrolyses triglycerides at the glycerol/fatty acid bond (primarily in positions 1 and 3) within the duodenum to yield mainly 2-monoglycerides, some diglycerides and free fatty acids2. The yielded glycerol can be taken up by the kidneys or the liver and converted into glucose through gluconeogenesis, thereby elevating the blood glucose level. Inhibition of lipase activity may reduce the rate of gluconeogenesis from the fat precursors and therefore aid in plummeting hyperglycaemia in diabetic patients. The CAQ and BAQ extracts of H. petiolare showed concentration-dependent lipase inhibitions (Figure 5), with the BAQ extract showing significantly higher inhibitory capacity compared to the standard acarbose. This further justifies the use of H. petiolare by the traditional healers in the treatment of diabetes and also corroborates the findings of Erasto, Adebola and Afolayan, (2011), and Mahop and Mayet, (2015), who listed H. petiolare amongst medicinal plants traditionally used in the treatment of diabetes mellitus9,23.\n\n\nConclusion\n\nAmong all the extracts used, BAQ and CAQ extracts have been shown in this study to have no apparent toxicity to myocytes and hepatocytes. This means either extract can be used in herbal treatment with minimal cytotoxicity on the liver and muscle cells. More studies, however, need to be done to elucidate the effect of the aqueous extracts of H. petiolare on the GLUT4 translocation cascade pathway. The whole plant BAQ extract of H. petiolare has the biggest effect on glucose uptake in HepG2 (C3A) hepatocytes, and the highest levels of α-amylase, α-glucosidase, and lipase inhibition. The aqueous extracts, more importantly the BAQ extract, may, therefore, contain pharmacologically active hypoglycaemic chemicals, which may be effective in the treatment of diabetes mellitus.\n\n\nData availability\n\nFigshare: In-vitro evaluation of the anti-diabetic potential of Helichrysum petiolare Hilliard & B.L. Burtt using HepG2 (C3A) and L6 cell lines. https://doi.org/10.6084/m9.figshare.1303500217.\n\nThis project contains the following underlying data:\n\n- A-Amylase.xlsx\n\n- A-Glucosidase Assay.xlsx\n\n- Glucose Utilization C3A.xlsx\n\n- Glucose Utilization L6.xlsx\n\n- Lipase Assay.xlsx\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgments\n\nThe authors wish to thank the National Research Foundation (NRF) of South Africa for funding this work, and also Professor C.N. Cupido for authenticating the plant used in this study.\n\n\nReferences\n\nSagbo IJ, van de Venter M, Koekemoer T, et al.: In Vitro Antidiabetic Activity and Mechanism of Action of Brachylaena elliptica (Thunb.) DC. Evid Based Complement Alternat Med. 2018; 2018: 4170372. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCrook MA: Clinical Biochemistry & Metabolic Medicine. 2012; 1–430. Reference Source\n\nMudumbi JBN, Ntwampe SKO, Mekuto L, et al.: The role of pollutants in type 2 diabetes mellitus (T2DM) and their prospective impact on phytomedicinal treatment strategies. Environ Monit Assess. 2018; 190(5): 262. PubMed Abstract \| Publisher Full Text\n\nBalogun FO, Tshabalala NT, Ashafa AOT, et al.: Antidiabetic Medicinal Plants Used by the Basotho Tribe of Eastern Free State: A Review. J Diabetes Res. 2016; 2016: 4602820. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAkinrinde A, Koekemoer T, Van De Venter M, et al.: In Vitro Investigation of Potential Anti-Diabetic Activity of the Corm Extract of Hypoxis Argentea Harv. Ex Baker. Acta Pharm. 2018; 68(4): 389–407. PubMed Abstract \| Publisher Full Text\n\nMohammed A, Ibrahim MA, Islam MS: African medicinal plants with antidiabetic potentials: A review. Planta Med. 2014; 80(5): 354–77. PubMed Abstract \| Publisher Full Text\n\nCragg GM, Newman DJ: Natural products: A continuing source of novel drug leads. Biochim Biophys Acta. 2013; 1830(6): 3670–95. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDay C: Traditional plant treatments for diabetes mellitus: pharmaceutical foods. Br J Nutr. 1998; 80(1): 5–6. PubMed Abstract \| Publisher Full Text\n\nErasto P, Adebola PO, Afolayan AJ: An ethnobotanical study of plants used for the treatment of diabetes in the Eastern Cape Province, South Africa. African J Biotechnol. 2011; 4(12): 1458–60. Reference Source\n\nOdeyemi S, Bradley G: Medicinal Plants Used for the Traditional Management of Diabetes in the Eastern Cape, South Africa: Pharmacology and Toxicology. Molecules. 2018; 23(11): 2759. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMaroyi A: Helichrysum Petiolare Hilliard and B. L. Burtt: A review of its medicinal uses, phytochemistry, and biological activities. Asian J Pharm Clin Res. 2019; 12(6): 69–71. Publisher Full Text\n\nKaleeswaran B, Ilavenil S, Ravikumar S: Screening of phytochemical properties and antibacterial activity of Cynodon dactylon L. Int J Curr Res. 2010; 3: 83–8. Reference Source\n\nMosmann T: Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods. 1983; 65(1–2): 55–63. PubMed Abstract \| Publisher Full Text\n\nvan de Venter M, Roux S, Bungu LC, et al.: Antidiabetic screening and scoring of 11 plants traditionally used in South Africa. J Ethnopharmacol. 2008; 119(1): 81–6. PubMed Abstract \| Publisher Full Text\n\nWickramaratne MN, Punchihewa JC, Wickramaratne DBM: In-vitro alpha amylase inhibitory activity of the leaf extracts of adenanthera pavonina. BMC Complement Altern Med. 2016; 16(1): 466. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJaradat N, Zaid A, Hussein F, et al.: Anti-Lipase Potential of the Organic and Aqueous Extracts of Ten Traditional Edible and Medicinal Plants in Palestine; a Comparison Study with Orlistat. Medicines (Basel). 2017; 4(4): 89. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAladejana AE, Bradley G, Afolayan AJ: In-vitro evaluation of the anti-diabetic potential of Helichrysum petiolare Hilliard & B.L. Burtt using HepG2 (C3A) and L6 cell lines. figshare. Dataset, 2020. http://www.doi.org/10.6084/m9.figshare.13035002.v1\n\nAladejana AE, Bradley G, Afolayan AJ: In-vitro evaluation of the cytotoxicity, mitotoxicity and lipotoxicity potentials of Helichrysum petiolare Hilliard & B.L. Burtt using HepG2 (C3A) Cell-lines. Med Plants - Int J Phytomedicines Relat Ind. 2020; 12(3): 1–8.\n\nLourens ACU, Van Vuuren SF, Viljoen AM, et al.: Antimicrobial activity and in vitro cytotoxicity of selected South African Helichrysum species. South African J Bot. 2011; 77(1): 229–35. Publisher Full Text\n\nAryaeian N, Sedehi SK, Arablou T: Polyphenols and their effects on diabetes management: A review. Med J Islam Repub Iran. 2017; 31: 134. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAladejana AE, Bradley G, Afolayan AJ: Phytochemical Screening and In-Vitro Antioxidant Activities of Various Extracts of Helichrysum petiolare Hilliard & B . L . Burtt used for the Treatment of Diabetes Mellitus in the Eastern Cape Province of South Africa. Int J Pharm Sci Res. 2020; 2020: 1-16 (in-press).\n\nHanhineva K, Törrönen R, Bondia-Pons I, et al.: Impact of Dietary Polyphenols on Carbohydrate Metabolism. 2010; 11(4): 1365–402. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMahop MT, Mayet M: En route to biopiracy? Ethnobotanical research on anti diabetic medicinal plants in the Eastern Cape Province, South Africa. African J Biotechnol. 2015; 6(June): 1684–5315." }	[ { "id": "79879", "date": "04 Mar 2021", "name": "Abimbola Oloye", "expertise": [ "Reviewer Expertise Reproductive toxicology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study underscores the importance of aqueous extract (Cold and Boiled) of Helichrysum petiolare Hilliard &B.L. Burtt in ameliorating Diabetes type 2 via enhanced glucose uptake and appreciable inhibition of α–amylase and α-glucosidase. Easy accessibility to the plant by locals and non toxic nature of the aqueous extract make this study pertinent and beneficial.\n\nI will like the authors to fill the following gaps:\nIn what form is the plant extract consumed by the locals. i.e what medium of extraction do they employ for consumption? If local gin (methanol or ethanol in some cases) is used, then the associated toxicity revealed in this work becomes a vital piece for dissemination to prevent risk to life.\n\nCan the authors add the Herberium number obtained for the plant to their report?\n\nThe percentage yield of extract from the oven dried plant should be presented. It is expected that the oven dried plant should have been weighed and the yield calculated after the extraction exercise.\n\nFinally, I will like to submit that the study was quite revealing. Further studies to unravel and elucidate the pharmacological active factors contained in the plant, which might be responsible for the anti-diabetic properties of the plant, will be highly beneficial.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "79881", "date": "08 Mar 2021", "name": "Yihong Bao", "expertise": [ "Reviewer Expertise Development and deep processing of forest resources", "enzyme engineering and biotechnology", "separation and application of natural active factors", "etc." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this work, the authors explored the anti-diabetic potentials of the ethanol, cold aqueous (CAQ) and boiled aqueous (BAQ) extracts of Helichrysum petiolare Hilliard & B.L. Burtt in vitro. They explored the utilization of glucose by L6 myocytes and HepG2 (C3A) hepatocytes under the intervention of different concentrations of extracts. In addition, they also evaluated the inhibitory effect of extracts on α-amylase, α-glucosidase and lipase.\nSpecific comments:\nThe Helichrysum petiolare in the keywords should be in italics.\n\nWhen preparing the ethanol extract, what is the concentration of ethanol?\n\nIn the section of \"Cell culture maintenance\", the growth medium is RPMI 1640. There are some contradictions.\n\n\"The blend was cooled down to optimum temperature and mixed with 5 ml of distilled water\". How to control to reach the \"optimal temperature\"?\n\nIn the section of ''α-glucosidase inhibition assay'', the article mentioned ''a blank'' and ''sample blank'', why these two groups of data are not reflected in the formula of %inhibition.\n\nThe author dissolved the extract with DMSO and determined the concentration. After that, the volume was adjusted to 1 mL with Tri-HCl. The concentration of the extract in the system has changed. Should the concentration of the extract be recalculated here?\n\nThe author consider that concentration-dependent increases in glucose uptake in the BAQ treated cells. However, there are only three concentrations of extracts, and there was no significant difference in the utilization of glucose between the 50 μg/ml and 100 μg/ml. Why the authors didn’t evaluate the inhibitory rate at other concentrations.\n\nThe abbreviation \"ETQ\" in the Figure does not appear in the text. It should be explained in the text, not just in the legend.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "6498", "date": "01 Apr 2021", "name": "Adebowale Emmanuel Aladejana", "role": "Author Response", "response": "Dear Yhiong Bao Below are responses to your 3rd, 5th, 6th, and 7th comments (response to other comments have been effected in the text): 3) We used a method described by Sagbo et al., (2018) and van de Venter et al., (2008), we will appreciate it if you elaborate on any contradiction you observed. 5) There was no need to include the sample blank (zero enzyme activity) in the equation since it was used to zero the spectrophotometer. 6) No, the enzyme was prepared using Tris-HCl and was then reacted with the extract. This has been corrected in the “pancreatic lipase inhibition” method section. 7) The statement “concentration-dependent increases in glucose uptake in the BAQ treated cells” was only made under “Glucose utilization in HepG2 (C3A) hepatocytes” (figure 2c), we believe you are referring to figure 1c under “Glucose utilization assay using L6 myocytes” where we have already stated that BAQ had no concentration-dependent effect on glucose uptake in the cells." } ] } ]	1	https://f1000research.com/articles/9-1240
https://f1000research.com/articles/8-1702/v1	30 Sep 19	{ "type": "Brief Report", "title": "A survey on the occurrence of Brachyspira pilosicoli and Brachyspira hyodysenteriae in growing-finishing pigs", "authors": [ "Arkadiusz Dors", "Ewelina Czyżewska-Dors", "Grzegorz Woźniakowski", "Ewelina Czyżewska-Dors", "Grzegorz Woźniakowski" ], "abstract": "Background: The major pathogenic intestinal spirochetes affecting pigs during the growing- finishing stage of production include Brachyspira hyodysenteriae and Brachyspira pilosicoli. Infections by these pathogens, which affect the economics of pig production, can result in mortality, growth rate losses and substantial antibiotic costs. The aim of this study was to assess the current occurrence of B. hyodysenteriae and B. pilosicoli in Polish pig herds. Moreover, associations between the presence of diarrhea or other intestinal pathogens and occurrence of B. hyodysenteriae and B. pilosicoli in pigs were investigated. Methods: Between January 2017 and August 2019, a total of 401 samples of pig feces from 95 different herds were submitted to the National Veterinary Research Institute of Poland. These samples were obtained from pigs older than 7 weeks. All the received fecal samples were examined for the presence of B. hyodysenteriae, B. pilosicoli and Lawsonia intracellularis by real-time PCR. Results: For B. pilosicoli, 4.5% (95% CI, 2.5–7.0%) of samples and 13.7% (95% CI, 7.5–22.3%) of herds were positive. Out of 12 samples, B. pilosicoli was detected simultaneously with L. intracellularis, B. hyodysenteriae and B. pilosicoli were detected alone in two samples each. In terms of B. hyodysenteriae, 7.0% of samples (95% CI, 4.7–9.9%) from 18.9% of herds (95% CI, 11.6–28.3%) were positive in real time PCR. The presence of B. hyodysenteriae in fecal samples was associated with the presence of diarrhea in pigs. Conclusions: This study confirmed that B. pilosicoli infections occur in Polish pig herds, but the prevalence is at a low level and the presence of B. pilosicoli is not associated with the development of diarrhea in pigs. B. hyodysenteriae is still a common cause of diarrhea among pigs from Polish herds.", "keywords": [ "Brachyspira pilosicoli", "Brachyspira hyodysenteriae", "Lawsonia intracellularis", "pigs", "intestinal pathogens", "enterocolitis", "diarrhea" ], "content": "Introduction\n\nThe question of routine surveillance to monitor Brachyspira species infections in pigs at local, national and international levels is addressed by experts and authorities (Hampson et al., 2015). The major pathogenic intestinal spirochetes affecting pigs during the growing-finishing stage of production include Brachyspira hyodysenteriae and Brachyspira pilosicoli. B. hyodysenteriae is the cause of swine dysentery (SD) – a severe, enteric disease of pigs characterized by mucohemorrhagic diarrhea and inflammation in the large intestine. B. hyodysenteriae is present worldwide and affects the economics of pig production, resulting in mortality, growth rate losses and substantial antibiotic costs. Another brachyspiral disease with mild colitis and diarrhea is porcine intestinal spirochetosis or porcine colonic spirochetosis (PIS/PCS). B. pilosicoli is the causative agent of PIS/PCS, a disease that implies an important economic cost resulting from reduced growth performance and poor feed conversion (Duhamel, 1998). Most Brachyspira species have a restricted host range, whereas B. pilosicoli colonizes a wide range of hosts, including humans, and has the potential for interspecies transmission. There is also a potential for zoonotic transmission, especially in places where animals and humans live in close proximity, or in people working with intensively farmed pigs or chickens, due to the increased risk of exposure. Some species of the genus Brachyspira, including B. pilosicoli, can cause the disease in humans. There are a few reports on B. pilosicoli-associated human intestinal spirochetosis (HIS) (Hampson, 2018). The subclinical colonization of pigs, with B. pilosicoli is not uncommon and has been detected in several farms (Biksi et al., 2007). On other farms, B. pilosicoli were isolated from diseased pigs as the only causative agent or simultaneously with other enteric pathogens as part of a mixed infection (Reiner et al., 2011; Stege et al., 2000).\n\nRecent changes in the management of pig farms and the movement of pigs within the EU have resulted in a shift in the relative prevalence of pathogenic Brachyspira species. There are very few studies addressing the prevalence of B. hyodysenteriae in pigs in Poland and only one concerning B. pilosicoli (Pławińska et al., 2004). The aim of the study was to assess the current occurrence of B. hyodysenteriae and B. pilosicoli in Polish pig herds. Moreover, associations between the presence of diarrhea or other intestinal pathogens and the occurrence of B. hyodysenteriae and B. pilosicoli in pigs were investigated.\n\n\nMethods\n\nFecal samples used in this study were submitted to the Department of Swine Diseases of the National Veterinary Research Institute (NVRI) for commercial laboratory diagnostics of selected porcine bacterial pathogens. Between January 2017 and August 2019, a total of 401 samples of pig feces were submitted to the NVRI. These samples originated from 95 different Polish pig herds, from pigs older than 7 weeks. All received fecal samples were submitted to the NVRI to be examined for the presence of B. hyodysenteriae and/or Lawsonia intracellularis. At that time, none of the diagnostic tools for B. pilosicoli identification were available for NVRI customers.\n\nOwing to differing reasons for testing submitted fecal samples, three groups were distinguished. The first group of samples were obtained from pigs subjected to routine monitoring of herds free of one or both of the aforementioned pathogens (B. hyodysenteriae, L. intracellularis). The second group was made up of samples from pigs with clinical sings of diarrhea, where B. hyodysenteriae or L. intracellularis was suspected to be a cause of disease. The last group of samples was submitted to the laboratory due to unrecognized pathogen status and a history of diarrhea in the herd.\n\nTotal genomic DNA was extracted from the fecal samples using a commercial isolation kit (Genomic Mini, A&A Biotechnology, Gdynia, Poland), according to manufacturer’s recommendations. Extracted DNA samples were stored at -20°C until examination. All samples were tested by separate singleplex real-time PCR assays for B. hyodysenteriae, B. pilosicoli and L. intracellularis according to the methods described previously (Ståhl et al., 2011; Zmudzki et al., 2012). Primers were obtained from a commercial source (Genomed S.A., Poland). The sequences of primers and probes are as follows: for B. hyodysenteriae (forward primer: 5'-TATGAAGAAGGCAGCAGACGTTTAT-3', reverse primer: 5'-GTAGGAAGAAGAAATCTGACAATGCA-3', probe: 5'-FAM-ACACAATCATGCTGAAGC-TAMRA-3') (Akase et al., 2009); for B. pilosicoli (forward primer: 5'-GTAGTCGATGGGAAACAGGT-3', reverse primer: 5'-TTACTCACCACAAGTCTCGG-3', probe: 5'-FAM-TATTCGACGAGGATAACCATCACCT-BHQ-1-3') (Ståhl et al., 2011); for L. intracellularis (forward primer: 5'-GCGCGCGTAGGTGGTTATAT-3', reverse primer: 5'-GCCACCCTCTCCGATACTCA-3', probe: 5'-FAM-CACCGCTTAACGGTGGAACAGCCTT-TAMRA-3’) (Lindecrona et al., 2002). All assays were carried out using the Rotor-Gene Q real-time PCR system (Qiagen, Hilden, Germany).\n\nReal-time PCR assays were run using a commercially available master mix Quantitect Probe PCR kit (Qiagen, Hilden, Germany). For B. hyodysenteriae, 12.5 μl of the master mix was combined with 0.5 μl of each primer, diluted to 20 μM and 0.5 μl of the probe, diluted to 20 μM and 6 μl of DNase-free water. The DNA template was added at 5 μl per reaction for a total reaction volume of 25 μl. PCR was run, as follows: 95°C for 15 mins, followed by 50 cycles at 95°C for 15 secs and 52°C for 1 min. For B. pilosicoli, 12.5 μl of the master mix was combined with 0.5 μl of each primer, diluted to 20 μM and 0.5 μl of the probe, diluted to 10 μM and 8 μl of DNase-free water. The DNA template was added at 3 μl per reaction for a total reaction volume of 25 μl. PCR was run as follows: 95°C for 15 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. For L. intracellularis, 12.5 μl of the master mix was combined with 0.5 μl of each primer, diluted to 20 μM and 0.5 μl of the probe, diluted to 20 μM and 6 μl of DNase-free water. The DNA template was added at 5 μl per reaction for a total reaction volume of 25 μl. PCR was run as follows: 95°C for 15 min, followed by 40 cycles of 95°C for 15 sec and 62°C for 1 min.\n\nA herd was defined as positive when at least one fecal sample taken from the herd had a positive PCR result. Percentages of positive samples/herds with a 95% two-sides exact binominal confidence interval (CI) were reported. Differences in the presence of pathogens between different fecal samples groups were established by a chi-square test (statistically significant at p < 0.05). Pairwise comparisons with Bonferroni corrections of the p-values were performed.\n\n\nResults\n\nAmong a total of 401 samples 218 were submitted to the NVRI laboratory for the routine monitoring of pig herds. Of these, 70 samples originated from pigs with the clinical manifestation of diarrhea. The remaining 113 samples originated from herds with a history of diarrhea but of an unknown status, in terms of Brachyspira spp. occurrence. Underlying data are available on figshare (Dors et al., 2019).\n\nThe overall occurrence of B. hyodysenteriae and B. pilosicoli in assessed pig herds in Poland is presented in Figure 1. Real-time PCR detected B. pilosicoli in 18 samples from pigs in 13 different herds. This means that 4.5% (95% CI, 2.5–7.0%) of samples and 13.7% (95% CI, 7.5–22.3%) of herds were positive for B. pilosicoli. Out of 12 samples, B. pilosicoli was detected simultaneously with L. intracellularis, B. hyodysenteriae and B. pilosicoli were detected alone in two samples each. In terms of B. hyodysenteriae, 7.0% of samples (95% CI, 4.7–9.9%) from 18.9% herds (95% CI, 11.6–28.3%) were positive using real-time PCR.\n\nDifferences in the presence of B. hyodysenteriae and B. pilosicoli in the fecal samples obtained from pigs with diarrhea, from apparently healthy pigs, but originating from herds with a history of diarrhea and from pigs undergoing routine monitoring from herds free of B. hyodysenteriae and/or L. intracellularis are shown in Table 1.\n\n*Statistically significant difference (p<0.05) between sample groups.\n\nAdditional analyses were completed to compare the influence of L. intracellularis infection and the presence of Brachyspira spp., in fecal samples. The occurrence of B. hyodysenteriae in pigs whose feces was confirmed to be positive for L. intracellularis was 7.9%, compared to 7.3% in pigs negative for L. intracellularis. However, considering the simultaneous occurrence of B. pilosicoli and L. intracellularis, we found that the percentage of samples positive for B. pilosicoli was significantly higher in pigs simultaneously infected by L. intracellularis (10.8%) compared to L. intracellularis-negative pigs (2.2%).\n\n\nDiscussion and conclusions\n\nThe results of this study confirm that B. pilosicoli infections occur in Polish pig herds. A previous study reported only one positive sample among 127 samples from 23 pig farms (Pławińska et al., 2004). Our results show that B. pilosicoli is present in Polish pig herds, but that the prevalence is low, reaching 13.7% of herds and 4.5% of samples. Notably, considerably higher prevalence of B. pilosicoli infection has been detected in other countries, such as Germany (31.6%, Reiner et al., 2011), Denmark (19%, Stege et al., 2000) and Hungary (61.3%, Biksi et al., 2007). Therefore, the targeted sampling of pigs from age groups in which detection of this pathogen is most likely and random selection of Polish pig herds is necessary to assess the true prevalence of B. pilosicoli.\n\nAn association between B. pilosicoli infections in pigs and the occurrence of diarrhea in this study was not confirmed. Our results are in line with some previous reports (Biksi et al., 2007; Weber et al., 2015), but other authors have demonstrated positive associations between presence of diarrhea and B. pilosicoli detection (Fellström et al., 1996; Stege et al., 2001). It seems that the subclinical colonization of pigs by B. pilosicoli is predominant in pigs, in Poland. Considering the causality of PIS/PCS, other factors causing the development of diarrhea in pigs, besides the B. pilosicoli infection, should be considered. B. pilosicoli colonization and/or disease expression can be influenced by diet (Hopwood et al., 2002; Stege et al., 2001). Moreover, concurrent infection can influence B. pilosicoli colonization and disease manifestation.\n\nIn our study, we have found that most of the positive samples came from pigs infected at the same time with L. intracellularis. Similar findings have been reported in previous studies (Biksi et al., 2007; Jacobson et al., 2003; Jacobson et al., 2005; Merialdi et al., 2003). Therefore, there is a need for further investigation to determine a risk factors and an association between the presence of B. pilosicoli in feces and the clinical signs or pig performance.\n\nThe occurrence of B. hyodysenteriae in our investigation was more common than B. pilosicoli and was higher than reported previously (Dors et al., 2015). Current results on the prevalence of B. hyodysenteriae could be biased, due to the large number of samples submitted to the NVRI with suspected clinical SD. Nonetheless, SD is still a common cause of diarrhea among pigs from Polish herds, despite improving biosecurity, management and disease control.\n\n\nData availability\n\nFigshare: A survey on the occurrence of Brachyspira pilosicoli and Brachyspira hyodysenteriae in growing-finishing pigs. https://doi.org/10.6084/m9.figshare.9878612.v1 (Dors et al., 2019).\n\nThis project contains data on detection of infection with each pathogen studied for each sample. 1, yes; 0, no.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgments\n\nWe wish to express our gratitude to the veterinary practitioners who supplied us with fecal samples.\n\n\nReferences\n\nAkase S, Uchitani Y, Sohmura Y, et al.: Application of real time PCR for diagnosis of Swine Dysentery. J Vet Med Sci. 2009; 71(3): 359–362. PubMed Abstract \| Publisher Full Text\n\nBiksi I, Lőrincz M, Molnár B, et al.: Prevalence of selected enteropathogenic bacteria in Hungarian finishing pigs. Acta Vet Hung. 2007; 55(2): 219–227. PubMed Abstract \| Publisher Full Text\n\nDors A, Czyżewska-Dors E, Woźniakowski G: A survey on the occurrence of Brachyspira pilosicoli and Brachyspira hyodysenteriae in growing-finishing pigs. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.9878612.v1\n\nDors A, Pomorska-Mól M, Czyżewska E, et al.: Prevalence and risk factors for Lawsonia intracellularis, Brachyspira hyodysenteriae and Salmonella spp. in finishing pigs in Polish farrow-to-finish swine herds. Pol J Vet Sci. 2015; 18(4): 825–831. PubMed Abstract \| Publisher Full Text\n\nDuhamel GE: Colonic spirochetosis caused by Serpulina pilosicoli. Vet Med Large Anim Prac. 1998; 19: 14–22.\n\nFellström C, Pettersson B, Johansson KE, et al.: Prevalence of Serpulina species in relation to diarrhea and feed medication in pig-rearing herds in Sweden. Am J Vet Res. 1996; 57(6): 807–811. PubMed Abstract\n\nHampson DJ: The Spirochete Brachyspira pilosicoli, Enteric Pathogen of Animals and Humans. Clin Microbiol Rev. 2018; 31(1): pii: e00087-17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHampson DJ, La T, Phillips ND: Emergence of Brachyspira species and strains: reinforcing the need for surveillance. Porcine Health Manag. 2015; 1: 8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHopwood DE, Pethick DW, Hampson DJ: Increasing the viscosity of the intestinal contents stimulates proliferation of enterotoxigenic Escherichia coli and Brachyspira pilosicoli in weaner pigs. Br J Nutr. 2002; 88(5): 523–532. PubMed Abstract \| Publisher Full Text\n\nJacobson M, Hård af Segerstad C, Gunnarsson A, et al.: Diarrhoea in the growing pig - a comparison of clinical, morphological and microbial findings between animals from good and poor performance herds. Res Vet Sci. 2003; 74(2): 163–169. PubMed Abstract \| Publisher Full Text\n\nJacobson M, Gerth Löfstedt M, Holmgren N, et al.: The prevalences of Brachyspira spp. and Lawsonia intracellularis in Swedish piglet producing herds and wild boar population. J Vet Med B Infect Dis Vet Public Health. 2005; 52(9): 386–391. PubMed Abstract \| Publisher Full Text\n\nLindecrona RH, Jensen TK, Andersen PH, et al.: Application of a 5' nuclease assay for detection of Lawsonia intracellularis in fecal samples from pigs. J Clin Microbiol. 2002; 40(3): 984–987. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMerialdi G, Bonilauri P, Granelli F, et al.: Bacterial pathogens in field cases of clinical colitis in growing and finishing pigs in Italy. Vet Rec. 2003; 153(17): 529–530. PubMed Abstract \| Publisher Full Text\n\nPławińska J, Jakubowski T, Rzewuska M, et al.: Occurrence of Lawsonia Intracellularis and Brachyspira spp. infection in swine suffering from diarrhoea. Pol J Vet Sci. 2004; 7(3): 203–206. PubMed Abstract\n\nReiner G, Hillen S, von Berg S, et al.: Analysis of bacterial load and prevalence of mixed infections with Lawsonia intracellularis, Brachyspira hyodysenteriae and/or Brachyspira pilosicoli in German pigs with diarrhoea. Berl Munch Tierarztl Wochenschr. 2011; 124(5–6): 236–241. PubMed Abstract \| Publisher Full Text\n\nStåhl M, Kokotovic B, Hjulsager CK, et al.: The use of quantitative PCR for identification and quantification of Brachyspira pilosicoli, Lawsonia intracellularis and Escherichia coli fimbrial types F4 and F18 in pig feces. Vet Microbiol. 2011; 151(3–4): 307–314. PubMed Abstract \| Publisher Full Text\n\nStege H, Jensen TK, Møller K, et al.: Risk factors for intestinal pathogens in Danish finishing pig herds. Prev Vet Med. 2001; 50(1–2): 153–164. PubMed Abstract \| Publisher Full Text\n\nStege H, Jensen TK, Møller K, et al.: Prevalence of intestinal pathogens in Danish finishing pig herds. Prev Vet Med. 2000; 46(4): 279–292. PubMed Abstract \| Publisher Full Text\n\nWeber N, Nielsen JP, Jakobsen AS, et al.: Occurrence of diarrhoea and intestinal pathogens in non-medicated nursery pigs. Acta Vet Scand. 2015; 57: 64. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZmudzki J, Szczotka A, Podgórska K, et al.: Application of real-time PCR for detection of Lawsonia intracellularis and Brachyspira hyodysenteriae in fecal samples from pigs. Pol J Vet Sci. 2012; 15(2): 267–273. PubMed Abstract \| Publisher Full Text" }	[ { "id": "55873", "date": "26 Nov 2019", "name": "Matheus costa", "expertise": [ "Reviewer Expertise Veterinary medicine", "swine medicine", "molecular diagnostic tests", "microbiome", "transcriptome." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI read with great interest this article, that sought to investigate the presence of Bhyo and Bpilo in polish herds. As the authors observed, this is a somewhat challenging goal: both bacterium can survive in the healthy host, thus it's hard to evaluate the meaning of their presence.\nMethods - Fecal samples - Please include a numeric description of each group. You mention the total number of fecal samples, but not how many came from X many herds, and how many are part of each \"submission group\".\nResults - How many samples were tested out of the 18 that were found positive for Bpilo? Please include the actual numbers in all your descriptions.\nFigure 1 - Please include actual n to the data shown. Also italicize scientific names.\nLawsonia comparison - Please include actual numbers in all the descriptions (n=?). A statistical test to show that samples positive for LI are more likely to be positive with Bpilo would be interesting here, besides the simple description.\n\nDiscussion - Was is thought that Poland was free of Bpilo? The first sentence is odd.\n\nPlease acknowledge that this data set is inherently biased (at least partially, except for the ones that are routine surveillance but we don't know how many samples were part of the group...).\n\nPlease include a conclusion statement, and clearly lay out your main findings.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5296", "date": "13 Mar 2020", "name": "arkadiusz dors", "role": "Author Response", "response": "I would like to begin by thanking the reviewer for the valuable comments. Below we have included our responses to specific comments.Methods - Fecal samples - Please include a numeric description of each group. You mention the total number of fecal samples, but not how many came from X many herds, and how many are part of each \"submission group\".Number of fecal samples in \"submission group\" was provided in results section but according to your suggestion we have number also in methods.Results - How many samples were tested out of the 18 that were found positive for Bpilo? Please include the actual numbers in all your descriptions.Changed as suggested by reviewer.Figure 1 - Please include actual n to the data shown. Also italicize scientific names.Figure 1 was removed because of repetition of results described in the text.Lawsonia comparison - Please include actual numbers in all the descriptions (n=?). A statistical test to show that samples positive for LI are more likely to be positive with Bpilo would be interesting here, besides the simple description. Corrected as suggested by reviewer. Necessary explanation has been added to Methods section and p-values were added in the Results.Discussion - Was is thought that Poland was free of Bpilo? The first sentence is odd. Sentence was rephrasedPlease acknowledge that this data set is inherently biased (at least partially, except for the ones that are routine surveillance but we don't know how many samples were part of the group...). In discussion we have mentioned that: “Current results on the prevalence of B. hyodysenteriae could be biased, due to the large number of samples submitted to the NVRI with suspected clinical SD.”Please include a conclusion statement, and clearly lay out your main findings.Final conclusions was added at the end of discussion" } ] }, { "id": "57051", "date": "05 Dec 2019", "name": "Roberto M.C. Guedes", "expertise": [ "Reviewer Expertise enteropathogens of pigs" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGeneral comments: This study describes the occurrence of pathogenic spirochetes for swine in Poland herds using routine diagnostic cases submitted to their reference laboratory. As a result, sampling was bias for a prevalence study, so the decision of just describe as occurrence was adequate. The data represents important information for Poland swine production, but not so relevant for the rest of the World. Figure 1 is illustrative but it is a repetition of the data that it is already stated in the text. It seems that qPCR for B. pilosicoli was not performed at the arrival of the sample, in contrast to qPCR for B. hyodysenteriae and L. intracellularis. So, when was the qPCR for B. pilosicoli performed?\n\nSpecific comments: It was very difficult to list the modification required in the text as it does not have the lines numbered.\nAbstract:\nResults: “…simultaneously with L. intracellularis. B. hyodysenteriae and B. pilosicoli were …”\nSuggestion of Key-words: pathogenic spirochetes, Lawsonia intracellularis, swine, intestinal pathogens, enterocolitis, diarrhea.\nResults:\nThe 4th and 5th sentences of the second paragraph are confusing. Rewrite. The 3rd paragraph is too long and confusing. Rewrite.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5297", "date": "13 Mar 2020", "name": "arkadiusz dors", "role": "Author Response", "response": "I would like to begin by thanking the reviewer for the valuable comments. Below we have included our responses to specific comments. Figure 1 is illustrative but it is a repetition of the data that it is already stated in the text.Figure 1 was removed because of repetition of results described in the text.It seems that qPCR for B. pilosicoli was not performed at the arrival of the sample, in contrast to qPCR for B. hyodysenteriae and L. intracellularis. So, when was the qPCR for B. pilosicoli performed?Adequate explanation was added to Methods section.Abstract: Results: “…simultaneously with L. intracellularis. B. hyodysenteriae and B. pilosicoli were …”Abstract was adjusted to all changes that were made within the article.Suggestion of Key-words: pathogenic spirochetes, Lawsonia intracellularis, swine, intestinal pathogens, enterocolitis, diarrhea.We found it difficult to change.Results: The 4th and 5th sentences of the second paragraph are confusing. Rewrite. The 3rd paragraph is too long and confusing. Rewrite.These sentences was rephrased according to reviewer suggestions." } ] } ]	1	https://f1000research.com/articles/8-1702
https://f1000research.com/articles/9-774/v1	27 Jul 20	{ "type": "Research Article", "title": "Extended spectrum β-lactamases and class C β-lactamases gene frequency in Pseudomonas aeruginosa isolated from various clinical specimens in Khartoum State, Sudan: a cross sectional study", "authors": [ "Dina N. Abdelrahman", "Aya A. Taha", "Mazar M. Dafaallah", "Alaa Abdelgafoor Mohammed", "Abdel Rahim M. El Hussein", "Ahmed I. Hashim", "Yousif F. Hamedelnil", "Hisham N. Altayb", "Aya A. Taha", "Mazar M. Dafaallah", "Alaa Abdelgafoor Mohammed", "Abdel Rahim M. El Hussein", "Ahmed I. Hashim", "Yousif F. Hamedelnil", "Hisham N. Altayb" ], "abstract": "Background: Pseudomonas aeruginosa is a pathogenic bacterium, causing nosocomial infections with intrinsic and acquired resistance mechanisms to a large group of antibiotics, including β-lactams. This study aimed to determine the susceptibility pattern to selected antibiotics and to index the first reported β-lactamases gene (extended spectrum β-lactamases (ESBLs) genes and class C β-lactamases genes) frequency in Ps. aeruginosa in Khartoum State, Sudan. Methods: 121 Ps. aeruginosa clinical isolates from various clinical specimens were used in this cross-sectional study conducted in Khartoum State. A total of 80 isolates were confirmed as Ps. aeruginosa through conventional identification methods and species-specific primers (the remaining 40 isolates were other bacterial species). The susceptibility pattern of the confirmed isolates to selected antibiotics was done following the Kirby Bauer disk diffusion method. Multiplex PCR was used for detection of seven β-lactamase genes (blaTEM, blaSHV, blaCTXM-1, blaVEB, blaOXA-1, blaAmpC and blaDHA). Results: Of the 80 confirmed Ps. aeruginosa isolates, 8 (10%) were resistant to Imipenem while all isolates were resistant to Amoxicillin and Amoxyclav (100%). A total of 43 (54%) Ps. aeruginosa isolates were positive for ESBLs genes, while 27 (34%) were positive for class C β-lactamases, and 20 (25%) were positive for both classes. Frequency of ESBLs genes was as follows: blaTEM, 19 (44.2%); blaSHV, 16 (37.2%); blaCTX-M1, 10 (23.3%); blaVEB, 14 (32.6%); and blaOXA-1, 7 (16.3%). Occurrence of class C β-lactamases genes was blaAmpC 22 (81.5%) and blaDHA 8 (29.6%). In total, 3 (11.1%) isolates were positive for both blaAmpC and blaDHA genes. Conclusion: Ps. aeruginosa isolates showed a high rate of β-lactamases production, with co-resistance to other antibiotic classes. The lowest resistance rate of Ps. aeruginosa was to Imipenem followed by Gentamicin and Ciprofloxacin. No statistically significant relationship between production of β-lactamases in Ps. aeruginosa and resistance to third generation cephalosporins was found.", "keywords": [ "ESBLs", "class C β-lactamase", "Polymerase Chain Reaction", "Ps. aeruginosa", "pyocyanin pigment", "Khartoum-Sudan." ], "content": "Introduction\n\nPseudomonas aeruginosa is one of the leading causes of nosocomial infections worldwide with high mortality rates, particularly among immunocompromised patients1. Ps. aeruginosa infections are difficult to treat, due to its extraordinary antimicrobial resistance to all available classes of antimicrobial agents2, including β-lactams, aminoglycosides and fluoroquinolones3. Resistance to β-lactams occurs by different mechanisms, including blaAmpC overexpression due to genetic mutations, mutant gene acquisition, overproduction of efflux system, or low permeability3. β-lactamases refer to enzymes that hydrolyze the amide bond of the β-lactam ring leading to drug inactivation and therapy failure4. β-lactamases are classified molecularly into four groups: class A (extended spectrum β-lactamases (ESBLs)), class B (metallo-β-lactamases), class C (cephalosporinases), and class D (oxacillinases)5.\n\nESBLs are enzymes that extend their hydrolyzing ability to hydrolyze broad spectrum cephalosporins6 and they also confer resistance to penicillins and narrow spectrum cephalosporins3. ESBLs are inhibited by β-lactamase inhibitors, such as clavulanic acid7. β-lactamases are transformed to ESBLs usually after point mutations in the β-lactamases gene. These mutations alter the substrate specificity because of changes in the amino acid sequences near the enzyme active site5. ESBLs producing Ps. aeruginosa have been reported worldwide in different countries8–17.\n\nAmpC β-lactamases are class C cephalosporinases that mediate bacterial resistance to cephalosporins and cephamycins. They also exhibit low rates of monobactam, cefepime and carbapenem hydrolysis18 and usually resist the inhibition by clavulanic acid4. Normally, AmpC is a chromosomal β-lactamase gene that is regulated by ampR gene and expressed constantly. Point mutations of ampR gene in Enterobacter cloacae activate AmpC that mediate resistance to β-lactams19. In Ps. aeruginosa over expressed AmpC β-lactamase mediate the resistance to broad spectrum cephalosporins3. AmpC β-lactamase in Ps. aeruginosa has also been reported in different countries around the world20–27.\n\nThe exact frequency of β-lactamase producing Ps. aeruginosa in Khartoum State, Sudan is unknown; therefore, the aim of this study was to determine the susceptibility pattern to selected antibiotics and to determine the frequency of β-lactamases producing Ps. areuginosa isolates collected in Khartoum State hospitals.\n\n\nMethods\n\nThis is a cross-sectional study conducted between February 2017 and October 2017. Ethical approval for the study was obtained from the ethical committee of the College of Medical Laboratory Science, Sudan University of Science and Technology (SUST) (ethical meeting no, SUST/DSR/1EC/EA2/2017; data, 07th January 2017). Written informed consent from participants was waived by the same ethical committee as the study only used previously collected human bio-specimens with limited participant data.\n\nA total of 121 clinical isolates, which initially identified as Ps. aeruginosa and bacteria other than Ps. aeruginosa was excluded. Selected isolates were obtained from Soba Teaching Hospital, Elribat University Hospital, National Laboratory for Public Health, Ear Nose Throat Hospital, and Military Hospital in Khartoum State. When there was a positive confirmation of Ps. aeruginosa, the study supervisor went and collected the sample from the hospital. The samples were collected from patients suffering from urinary tract infections, respiratory tract infections, blood infections, and wound and ear infections. Data pertaining to the site of infection was collected from hospital records. The bacteria were preserved in 20% glycerol and peptone water and stored at -20°C\n\nPhenotypic identity of the isolates was confirmed through conventional bacterial identification methods, such as Gram stain, oxidase test, and reactions in media containing sugars, such as Kligler Iron Agar, urease test and, citrate test. Pigment production was assessed using Muller Hinton agar, and then phenotypic identity confirmed by genotypic characterization using multiplex PCR, as previously described1.\n\nMuller Hinton medium (HiMedia, India) was prepared and sterilized as instructed by the manufacturer. Antimicrobial susceptibility testing was performed following the modified Kirby-Bauer disc diffusion method1 and the results interpreted according to Clinical Laboratory Standards Institute guidelines (CLSI, 2007). The following antimicrobial discs (HiMedia, India) were used for sensitivity testing: Amoxicillin (25 µg), Cefotaxime (30 µg), Amoxicillin-Clavulanic acid (30 µg), Gentamicin (10 µg), Ciprofloxacin (5 µg), Chloramphenicol (30 µg) and Imipenem (10 µg). Ps. aeruginosa ATCC 27853 was used as quality control strain to control the performance of the test and ensure that the test is properly performed.\n\nPhenotypic identity of the isolates was confirmed through conventional bacterial identification methods such as Gram stain, oxidase test, reactions in media containing sugars such as Kligler Iron Agar, urease test and citrate test.\n\nGenomic DNA was extracted by simple boiling method18. The extracted DNA was used as a template for amplification of target genes using multiplex PCR using TECHNE TC-312 (UK) thermocycler. Firstly, oprI and oprL primers (Table 1) (Macrogen, Korea) were used to confirm the identification of Ps. aeruginosa. Seven primer pairs (Table 1) (Macrogen, Korea) were used for detection of ESBLs genes (blaTEM, blaSHV, blaCTXM-1, blaVEB, blaOXA-1) and class C genes (blaAmpC and blaDHA). The oprI and oprL reaction was carried out with the following cycling conditions: denaturation at 95°C for 5 min; 33 cycles of denaturation at 95°C for 30 secs, annealing at 58°C for 30 sec and extension at 72°C for 30 secs, and final extension at 72°C for 5 min28.\n\nβ-lactamases detection was done in two batches; the first batch was used for detection of blaTEM, blaSHV, blaCTXM-1, blaAmpC and blaDHA, while the second batch was used for detection of blaVEB and blaOXA-1 genes. The first batch detection was done in 20 µl of final reaction mixture using Maxime PCR Premix kits (iNtRON Biotechnology, Korea), containing 13 µl of double distilled water (DDW), 0.3µl of each five forward and 0.3 µl of each five reverse primers (1.5 µl), 2 µl of Dimethyl sulfoxide (DMSO) and 2 µl of template DNA.\n\nAmplification of the second batch was done in 20µl using Maxime PCR Premix kits (iNtRON Biotechnology, Korea), containing 14.4 µl of DDW, 0.4 µl of each two forward and 0.4 µl of each two reverse primers (0.8 µl), 2 µl of DMSO and 2 µl of template DNA. Cycling conditions for both amplification reactions were as follows: initial denaturation at 94°C for 2 minutes, then 35 cycles of denaturation at 94°C for 30 secs, annealing at 54°C for 30 secs and extension at 72°C for 50 secs, and final extension at 72° for 5 minutes.\n\nAmplified products were analyzed by electrophoreses at 80 volts for 20 minutes on 1.5% agarose gel containing ethidium bromide and then visualized using UV transilluminator (Uvitec–UK) with 50bp or 100bp molecular DNA ladder (iNtRON Biotechnology, Korea).\n\nStatistical analysis of the data was performed using chi-square test (level of significance was 0.05) with SPSS software version 20 and GraphPad prism 5 demo.\n\n\nResults\n\nFrom 121 clinical isolates collected from different hospitals in Khartoum State, only 80 (66%) were confirmed as Ps. aeruginosa through conventional methods and species-specific primers; the remaining 41 (34%) isolates were considered as other Gram-negative rod bacteria. The distribution of clinical isolates according to site of infection was as follows: urine, 34 (42%); wound swab, 24 (30%); ear swab, 8 (10%); sputum, 8 (10%); and blood, 6 (7.5%).\n\nThe results of antimicrobial susceptibility test for selected antibiotics are presented in Table 2.\n\nOut of 80 Ps. aeruginosa isolates, 54 (68%) were Pyocyanin pigment producers, while 26 (32%) were not pigment producers. There was a significant association between pyocyanin pigment production and site of infection (P=0.000) (Figure 1). There was also a significant association between pigment production and resistance to Chloramphenicol (P=0.020) and Cefotaxime (P=0.000), while there was insignificant association between pigment production and resistance to other antimicrobials used in this study (Figure 2).\n\nMolecular detection of ESBLs showed that 43 (54%) of the isolates were positive for at least one ESBLs gene, while 37 (46%) were negative for all genes (Figure 3). The frequency of ESBLs gene presence among Ps. aeruginosa was as follows: blaTEM, 19 (44.2%); blaSHV, 16 (37.2%); blaCTX-M1, 10 (23.3%); blaVEB, 14 (32.6%); and blaOXA-1, 7 (16.3%). There was a significant association between the presence of ESBL genes in Ps. aeruginosa and site of infection (P=0.030) (Figure 3). Co-presence of more than one ESBLs gene among Ps. aeruginosa clinical isolates is presented in Table 3.\n\nClass C β-lactamases gene were positive in 27 (34%) Ps. aeruginosa isolates; while 53 (66%) were negative (Figure 4). The frequency of class C β-lactamases genes was as follows: blaAmpC, 22 (81.5%); blaDHA, 8 (29.6%); and 3 (11.1%) isolates were positive for both genes. Association between presence of Class C β-lactamases genes and site of infection was insignificant (P=0.215) (Figure 4).\n\nIn total, 25% of Ps. aeruginosa isolates were positive for both ESBLs and class C β-lactamase genes (Figure 5). Co-presence of ESBLs genes and class C β-lactamase among Ps. aeruginosa clinical isolates is presented in Table 4.\n\nNumber of genes per isolate*\n\n\nDiscussion\n\nPseudomonas aeruginosa is one of the main causative agents of serious nosocomial infections with increased reports of β-lactams resistant strains that makes treatment difficult and complicated19. Production of β-lactamases is one of the most common mechanisms of β-lactam resistance36.\n\nIn this study the frequency of ESBL genes among Ps. aeruginosa isolates was 53.8%. This frequency is close to a report from India by Qureshi and Bhatnagar (2016)37, where the frequency of ESBLs in Ps. aeruginosa isolates was 46%. However, the results of this study disagree with another report from India by Gupta et al. (2016)38 where the frequency of ESBLs in Ps. aeruginosa isolates was 22.9%. These discrepancies could be due to differences in strains of the clinical isolates, the antibiotics used or sample size.\n\nThe most abundant gene among the ESBLs producing Ps. aeruginosa isolates detected in this study was blaTEM gene (19, 44.2%), followed by blaSHV, blaVEB, blaCTXM-1 and blaOXA-1 (16 (37.2%), 14 (32.6%), 10 (23.3%) and 7 (16.3%), respectively). Similar results were reported by Salah et al. (2016)39 in Egypt concerning the presence of blaTEM, blaSHV and blaOXA-1 (50%, 33% and 17%, respectively). The frequency of blaTEM gene is also similar to that reported by Rafiee et al. (2014)40, which was present in 39.2% of isolates. In a study in Iran by Sales et al. (2017)41, similar results were reported concerning the presence of blaCTXM-1 (27.3%), while in India Jamali et al. (2017)15 reported a higher frequency (57.5%) of this gene.\n\nOn the other hand, the frequency of blaVEB and blaSHV genes in this study differs from that reported in Iran by Bokaeian et al. (2014)42 where blaVEB gene frequency was 13.3%, while the frequency of blaSHV gene was 6.6%. The report by Jamali et al. (2017)15 concerning the genes blaTEM (15%) and blaSHV (75%) are also different from those reported in this study, and this could be due to the variation in strains of clinical isolates and the sample size used.\n\nIn this study, the frequency of class C β-lactamase genes in Ps. aeruginosa isolates was 27 (34%). This result is close to a report from India by Gupta et al. (2016)38 where 43% of Ps. aeruginosa were AmpC producers, and disagrees with a report from Thailand by Katvoravutthichai et al. (2016)43 where 11% of Ps. aeruginosa isolates were AmpC producers. In this study, out of the class C β- lactamase producing Ps. aeruginosa isolates, 22 (81.5%) and 8 (29.6%) isolates were positive for blaAmpC and blaDHA genes, respectively. Qureshi and Bhatnagar (2016)37 in India reported that no Ps. aeruginosa isolates were positive for blaAmpC gene, while Rafiee et al. (2014)40 in Iran reported that 60.8% of Ps. aeruginosa were positive for blaAmpC gene.\n\nAll Ps. aeruginosa clinical isolates tested in our study were resistant to Amoxicillin and Amoxyclav and this may be due to the misuse of antibiotics in Sudan44, where plenty of antimicrobial agents are sold over the counter. This rate of resistance is higher than the rate of resistance reported by Ahmad et al. (2016)45 in Pakistan where the resistance to Amoxicillin and Amoxyclav was 73.4% and 67.7% respectively, and this could be justified by the time difference between the studies, as well as the difference in the strains and antibiotics used.\n\nThe resistance rate of Ps. aeruginosa to Imipenem was 10% (n=8). This may be due to the infrequent use of Imipenem antibiotics. This percentage agrees with a study reported in Pakistan by Ahmad et al. (2016)45 where 11.1% of Ps. aeruginosa isolates were resistant to Imipenem, while in Sudan Altom and Ahmed (2015)46 reported that 5.7% Ps. aeruginosa isolates were resistant to Imipenem. This finding may indicate that carbapenem resistance is on the rise in Ps. aeruginosa isolates from Sudan.\n\nIn this study, the number of Ps. aeruginosa isolates resistant to Cefotaxime, Chloramphenicol and Ceftazidime was 71 (88.8%), 71 (88.8%) and 46 (57.5%), respectively. The rate of resistance to Cefotaxime in this study is different from that reported in Pakistan by Ahmad et al. (2016)45 who found that 20.3% of Ps. aeruginosa isolates were resistant to Ceftazidime. The resistance rate in this study also disagrees with that reported by Albadawi (2010)47 in Sudan who found that resistance of Ps. aeruginosa to Ceftazidime and Cefotaxime were 31% and 42%, respectively. These findings also indicate the rapidly increasing rates of Ps. aeruginosa resistance to antimicrobial agents in Sudan perhaps due to antibiotic misuse.\n\nIn this study, 23 (28.8%) and 19 (23.8%) of Ps. aeruginosa clinical isolates were resistant to Ciprofloxacin and Gentamicin, respectively. Altom and Ahmed (2015)46 in Sudan also reported that 18.6% of Ps. aeruginosa were resistant to Gentamicin. Different results were reported by Ahmad et al. (2016)45, where the percentage of resistance to Gentamicin and Ciprofloxacin were 74.3% and 44%, respectively. These percentages are much higher than those reported in this study probably due to their different geographical location, study time difference and the antibiotic-use rates.\n\nIn this study, there was significant association between ESBLs production and site of infection (P=0.030). Ps. aeruginosa isolated from blood showed the highest ESBLs production followed by ear swab, urine, sputum and wound swab. This result agrees with a study in India reported by Basak et al. (2012)48 where the highest ESBLs producing Ps. aeruginosa isolates were from blood, but disagrees with Azizi et al. (2015)49 in Iran who found that highest ESBLs production was in Ps. aeruginosa isolated from wound followed by urine, sputum and blood. There was insignificant association between the presence of class C β-lactamase genes and site of infection (P=0.215) found in the present study. The highest frequency of class C β-lactamases genes in Ps. aeruginosa were isolated from blood followed by urine, ear swab, wound swab and sputum. These results agree with the study in India reported by Basak et al. (2012)48 in that the highest percentage of class C β-lactamase genes in Ps. aeruginosa were found in blood.\n\nIn the present study, there were four clinical isolates phenotypically sensitive to third generation cephalosporins (Ceftazidime and Cefotaxime) and genotypically positive for ESBLs genes. This result indicates that Ps. aeruginosa may carry hidden unexpressed genes that could be detected through molecular techniques. This result agrees with a study in India reported by Bajpai et al. (2017)50 where out of 38 phenotypically ESBL-negative isolates, 20 isolates were positive for ESBLs genes.\n\nIn this study, the frequency of pigment producing Ps. aeruginosa isolates was 67.5%. In a study in India a higher percentage was reported where 82.5% of Ps. aeruginosa were pigment producers (Finlayson and Brown, 2011)51. The present study revealed that there was no relationship between pigment production and pattern of antimicrobial resistance, except in Chloramphenicol (P=0.02) and Cefotaxime (P=0.00).\n\nIn this study, the relationship between pigment production and site of infection was significant (P=0.000). Ps. aeruginosa isolated from wound infections were the highest pigment producing isolates followed by isolates from blood, urine and sputum. There was no pigment production in Ps. aeruginosa isolated from ear infections in this study.\n\nThe variations between the results of this study and other reports could be attributed to the difference in antibiotic usage patterns in each region, economical causes, geographical differences, sample size, differences in time in which the studies were performed, and study population. Despite the significance of the present study, there were limitations that should be avoided in future studies, such as the small sample size, phenotypic detection of β-lactamases, coverage of other β-lactamase classes, and gene sequencing should be done in order to confirm and to identify all the genes that are carried by Ps. aeruginosa strains in Sudan.\n\n\nConclusion\n\nThis is study is of great importance as it raises attention to the existing problem of resistance to β-lactams in Ps. aeruginosa in Sudan. This study confirms the reports that a number of antibiotics are becoming useless for treating this problematic bacterium, since all the strains of Ps. aeruginosa isolates in this study were resistant to Amoxicillin and Amoxyclav. The best antibiotic sensitivity results obtained in this study were those of Imipenem, followed by Gentamicin and Ciprofloxacin. Moreover, Ps. aeruginosa isolates showed an increased rate of β-lactamase production with co-resistance with other classes of antibiotics. Of interest is the finding that clinical isolates were resistant phenotypically in high frequencies to Amoxicillin, Amoxyclav and a third generation antibiotic, Cephalosporin, and showed negative results genotypically, indicating that resistance to this family of antibiotics also exist by resistance mechanisms other than β-lactamases production. Also, our PCR results revealed that Ps. aeruginosa possesses hidden β-lactamases genes that can’t be detected phenotypically. Finally, this study highlighted for the first time the problem of misidentification of Ps. aeruginosa and other microorganisms in Khartoum hospitals as only 80 out of the 120 alleged isolates were confirmed to be Ps. aeruginosa through PCR.\n\n\nData availability\n\nFigshare: SPSS, https://doi.org/10.6084/m9.figshare.12453287.v251\n\nThis project contains the following underlying data:\n\nSPSS. sav (Result sheet for beta-lactamases detection and sensitivity testing)\n\nData Dictionary. docx\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nDeep thanks to all the Microbiology Department team at Sudan University of Science and Technology for their significant help during the study. 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PubMed Abstract\n\nMagoué CL, Melin P, Gangoue-Pieboji J, et al.: Prevalence and spread of extended-spectrum β-lactamase-producing Enterobacteriaceae in Ngaoundere, Cameroon. Clin Microbiol Infect. 2013; 19(9): E416–E20. PubMed Abstract \| Publisher Full Text\n\nAltayb HN, Siddig MA, El Amin NM, et al.: Molecular Characterization of CTX-M ESBLs among Pathogenic Enterobacteriaceae isolated from different regions in Sudan. 2018. Reference Source\n\nQureshi MA, Bhatnagar RK: Phenotypic Characterization of ESBL, AmpC and MBL Producers among the Clinical Isolates of Multidrug Resistant Pseudomonas aeruginosa. Int J Curr Microbiol App Sci. 2016; 5(10): 749–58. Publisher Full Text\n\nGupta R, Malik A, Rizvi M, et al.: Incidence of multidrug-resistant Pseudomonas spp. In ICU patients with special reference to ESBL, AMPC, MBL and biofilm production. J Glob Infect Dis. 2016; 8(1): 25–31. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSalah M, Azab M, Halaby H, et al.: Mutations in β-lactamases detected in multidrug resistant gram negative bacteria isolated from community acquired urinary tract infections in Assiut, Egypt. Afr J Microbiol Res. 2016; 10(46): 1938–43. Publisher Full Text\n\nRafiee R, Eftekhar F, Tabatabaei SA, et al.: Prevalence of extended-spectrum and metallo β-lactamase production in AmpC β-lactamase producing Pseudomonas aeruginosa isolates from burns. Jundishapur J Microbiol. 2014; 7(9): e16436. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSales A, Fathi R, Mobaiyen H: Molecular Study of the Prevalence of CTX-M1, CTX-M2, CTXM3 in Pseudomonas aeruginosa Isolated from Clinical Samples in Tabriz Town, Iran. Electronic J Biol. 2017; 13(3): 253–259. Reference Source\n\nBokaeian M, Zahedani SS, Bajgiran MS, et al.: Frequency of PER, VEB, SHV, TEM and CTX-M genes in resistant strains of Pseudomonas aeruginosa producing extended spectrum β-lactamases. Jundishapur J Microbiol. 2014; 8(1): e13783. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKatvoravutthichai C, Boonbumrung K, Tiyawisutsri R: Prevalence of β-lactamase classes A, C, and D among clinical isolates of Pseudomonas aeruginosa from a tertiary-level hospital in Bangkok, Thailand. Genet Mol Res. 2016; 15(3). PubMed Abstract \| Publisher Full Text\n\nAwad A, Eltayeb I, Baraka O: Changing antibiotics prescribing practices in health centers of Khartoum State, Sudan. Eur J Clin Pharmacol. 2006; 62(2): 135–42. PubMed Abstract \| Publisher Full Text\n\nAhmad M, Hassan M, Khalid A, et al.: Prevalence of extended spectrum β-lactamase and antimicrobial susceptibility pattern of clinical isolates of Pseudomonas from patients of Khyber Pakhtunkhwa, Pakistan. Biomed Res Int. 2016; 2016: 6068429. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAltom RB, Ahmed ST: Antimicrobial Susceptibility of Clinical Isolates of Pseudomonas aeruginosa to Aminoglycosides and Imipenem. B. Sc dissertation. Sudan University of Science and Technology. 2015.\n\nAlbadawi SA: Determination of Antimicrobial Susceptibility of Pseudomonas aeruginosa Isolated from Sudanese Patients. Post graduate Diploma dissertation. Sudan University of Science and Technology. 2010.\n\nBasak S, Attal RO, Rajurkar M: Pseudomonas aeruginosa and newer β-lactamases: an emerging resistance threat. Infection Control-Updates: InTech. 2012. Publisher Full Text\n\nAzizi H, Kheirandish F, Mirzaei M: Frequency of Extended-Spectrum Beta-Lactamase (ESBLs)-Multidrug Resistance Produced by Pseudomonas aeruginosa Isolated from Clinical Specimens in Khorramabad City, Iran. Br Microbiol Res J. 2015; 5(6): 490. Publisher Full Text\n\nBajpai T, Pandey M, Varma M, et al.: Prevalence of TEM, SHV, and CTX-M Beta-Lactamase genes in the urinary isolates of a tertiary care hospital. Avicenna J Med. 2017; 7(1): 12–16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbdelrahman DN, Taha AA, Dafaallah MM, et al.: SPSS. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12453287.v2" }	[ { "id": "68073", "date": "26 Aug 2020", "name": "Nobumichi Kobayashi", "expertise": [ "Reviewer Expertise microbiology", "public health" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a kind of primitive study that provides only limited information. Only importance is that this study was done in Sudan. However, still number of isolates were low, and errors in descriptions of technical terms are found in this manuscript.\nAbstract: Number of P. aeruginosa should be written as \"80\". \"121\" should not appear here, because 41 isolates were not P. aeruginosa.\n\n\"Ps. aeruginosa\" should be corrected as \"P. aeruginosa\", as formal abbreviation.\n\nAmoxyclav may be a commercial name of the drug. It should be written as general name. Maybe it is \"amoxicillin/clavulanate\".\n\nAuthors may have serious misunderstanding regarding the term of ESBL. NOT all of TEM, SHV, OXA type beta-lactamases are ESBL. TEM-1, 2, SHV-1, OXA-1 are not ESBL. Other subtypes of TEM, SHV, and OXA are called as \"ESBL\". Those subtypes can be identified only by sequencing of full-length ORF of these genes. In this manuscript, only CTX-M type gene can be called as \"ESBL\". However, it is not sure that TEM, SHV, OXA type genes are ESBL genes or not, because sequence was not determined. In this regard, this manuscript must be revised substantially. Authors can mention CTX-M as ESBL, others should not be referred as ESBL, but just individual enzyme nams, e.g., \"TEM gene\". According to this alteration, whole manuscript should be rewitten.\n\nCTX-M-1 should be written as \"CTX-M-1 group\". CTX-M types cannot be determined by only PCR. Authors should read and study literature on ESBL and betalactamases of bacteria, to exactly understand their nomenclature and definition.\n\nTable 3 cannot be understood. Authors should make a table to show combination pattern of beta-lactamases and their frequency.\n\nFigure 5: \"Class AmpC genes\" is wrong. It should be \"Class C beta-lactamase\" or \"AmpC gene\". In this case, \"AmpC gene\" is better.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5911", "date": "15 Sep 2020", "name": "Dina Naser", "role": "Author Response", "response": "Classification of bla TEM, bla SHV, bla CTXM-1, bla VEB and bla OXA-1 changed from ESBLs to β-lactamases enzymes generally, they named in the draft as individual genes and classified as β-lactamases group. This change applied in the whole draft including tables and figures, even the title changed to “β-lactamases (bla TEM, bla SHV, bla CTXM-1, bla VEB, bla OXA-1) and class C β-lactamases gene frequency in Pseudomonas aeruginosa isolated from various clinical specimens in Khartoum State, Sudan: a cross sectional study” In abstract: The sample size mentioned as the confirmed 80 P. aeruginosa isolates, the remaining 41 isolate other than P. aeruginosa deleted. All “Ps. aeruginosa” in the text changed to “P. aeruginosa” as formal abbreviation. All “Amoxyclav” in the text changed to “amoxicillin/clavulanate” \"Class AmpC genes\" in figure 5 changed to “Class C β -lactamase” “ESBLs” changed to “β- lactamases” in figure 3 and figure 5." } ] } ]	1	https://f1000research.com/articles/9-774
https://f1000research.com/articles/10-268/v1	01 Apr 21	{ "type": "Opinion Article", "title": "Recommendations for the FAIRification of genomic track metadata", "authors": [ "Sveinung Gundersen", "Sanjay Boddu", "Salvador Capella-Gutierrez", "Finn Drabløs", "José M. Fernández", "Radmila Kompova", "Kieron Taylor", "Dmytro Titov", "Daniel Zerbino", "Eivind Hovig", "Sveinung Gundersen", "Sanjay Boddu", "Salvador Capella-Gutierrez", "Finn Drabløs", "José M. Fernández", "Radmila Kompova", "Kieron Taylor", "Dmytro Titov", "Daniel Zerbino" ], "abstract": "Background: Many types of data from genomic analyses can be represented as genomic tracks, i.e. features linked to the genomic coordinates of a reference genome. Examples of such data are epigenetic DNA methylation data, ChIP-seq peaks, germline or somatic DNA variants, as well as RNA-seq expression levels. Researchers often face difficulties in locating, accessing and combining relevant tracks from external sources, as well as locating the raw data, reducing the value of the generated information. Description of work: We propose to advance the application of FAIR data principles (Findable, Accessible, Interoperable, and Reusable) to produce searchable metadata for genomic tracks. Findability and Accessibility of metadata can then be ensured by a track search service that integrates globally identifiable metadata from various track hubs in the Track Hub Registry and other relevant repositories. Interoperability and Reusability need to be ensured by the specification and implementation of a basic set of recommendations for metadata. We have tested this concept by developing such a specification in a JSON Schema, called FAIRtracks, and have integrated it into a novel track search service, called TrackFind. We demonstrate practical usage by importing datasets through TrackFind into existing examples of relevant analytical tools for genomic tracks: EPICO and the GSuite HyperBrowser. Conclusion: We here provide a first iteration of a draft standard for genomic track metadata, as well as the accompanying software ecosystem. It can easily be adapted or extended to future needs of the research community regarding data, methods and tools, balancing the requirements of both data submitters and analytical end-users.", "keywords": [ "FAIR", "functional genomics", "epigenomics", "genomics", "metadata", "interoperability", "genomic tracks", "sequence annotations" ], "content": "Introduction\n\nGenomic track files were originally designed and optimised to be displayed within genomic web browsers, but have gradually become a de facto standard to store, distribute and analyse genome-wide datasets, mainly because of their efficient compression and indexing utilities. Many bioinformatics analyses are now being distributed, either privately or publicly, using such files. They are still predominantly used for graphical display, but can also be queried by statistical analysis tools, such as the GSuite HyperBrowser1, EPICO2, DeepBlue3 or the IHEC DataPortal4.\n\nFile formats used to represent tracks were not designed with FAIR data principles5 in mind, in particular with respect to metadata. Thus, their potential impact through re-use is greatly limited. Without proper metadata, an understanding of the protocol through which a track file was generated requires intensive literature curation and/or personal communication with the data generators, and these approaches are hampering usage. Further, if the exact provenance of a track file is not well understood, any downstream re-analysis is prey to artefacts. Thus, poorly annotated track files are rendered virtually useless for automated re-use.\n\nFurthermore, no central repository exists dedicated to storing track files and curated metadata. Instead, the track repositories are divided by species (mainly human vs. other systems) and domain (e.g., epigenomics, cancer, common variants, or rare-disease variants), with data portals typically being created during the life cycle of larger consortium undertakings (e.g., ENCODE or ICGC). The Track Hub Registry is a global centralised collection of public track hubs, and includes the metadata for a diverse collection of genomic tracks. However, the metadata content in the Track Hub Registry is not curated, and search facets are limited to high-level trackhub attributes.\n\nSignificant investments have gone into the generation of genomic tracks both within large consortia and independent groups, but the metadata is fragmented and disparate. Currently, significant legwork is required in order to identify, collect and consolidate a set of tracks to be used for a given research project. This is due to the often low quality of metadata annotations, like erroneous and missing metadata, duplicate attributes and/or records. More systematic deficiencies include difficulties accessing metadata and data (using various APIs and ad hoc scripts), and even a lack of adherence to the established formats for data and metadata. There is also a lack of standard metadata attributes across repositories and/or data types for simple filtering tasks. Critical information in need of standardization include the cell or tissue type of the sample, the assay type and main target of the experiment (if any), information on methods used to generate the track, as well as properties of the track files themselves such as the genome assembly version used6, the level and type of data condensation, and the geometric properties of the track7. Further, in many settings, manual interpretation of the metadata may be demanding, as non-standard terms are used, causing significant confusion and uncertainty, which results in a lack of trust in the data.\n\nWith the use of sufficient standards for metadata, and with matching development of functionality to make genomic tracks reusable, it is conceivable that one could mobilize all the available datasets world-wide relevant to a research question. This would positively impact both cost-efficiency by reuse of existing data as well as contribute to the evaluation of available datasets prior to the design of new projects. Ideally, all useful data should be easily searchable across repositories, downloadable, interoperable in terms of widely accepted formats, and directly usable by research software. Another formulation of this concept would be through the FAIR terms5: Findable, e.g., implementing metadata standards across track repositories; Accessible, e.g., making use of existing open protocols for accessing both data and metadata, even in the light of controlled access; Interoperable, e.g., utilizing open, well-defined and usable formats and protocols for track data; and Reusable, e.g., by describing data with sufficient attributes, such as provenance and usage policy, for reuse through a well-integrated ecosystem of functions across different fields of research.\n\nBased on our work in the ELIXIR Implementation Study on FAIRification of genomic tracks, we describe our efforts towards enabling genomic track FAIRness, through a description of a draft genomic track metadata standard, the development of a search function, and a demonstration of how interoperability and reuse can be achieved for the end-user by its inclusion in larger track registries.\n\nDomain-specific metadata standards exist that guide current efforts to annotate genomic datasets (see Table 1 for a summary of object types across these data models and their required attributes). The “International Nucleotide Sequence Database Collaboration” (INSDC) deposition model was developed jointly by NCBI, EMBL-EBI and DDBJ to facilitate genomic data exchangei,ii, and serves as a model for many of the standards described below. In particular, each data file is assigned to an “Experiment”, which links out to a specific “Study” and a specific “Sample”.\n\nA number of international efforts exist to extend the INSDC submission model to specific domains and/or organisms. For instance, IHEC8 is an umbrella organisation that brings together large epigenomic data production efforts, such as BLUEPRINT9, ENCODE10, and other large-scale initiatives. Each separate initiative produces tracks and deposits their data separately. However, IHEC brings together their metadata to a central location, so as to facilitate integrative analysis. Its data model is an extension of the INSDC submission model, to which attributes have been added, and where specific ontologies are recommendediiii.\n\n“Functional annotation of animal genomes” (FAANG)iv was created to coordinate the collection of functional genomics data (in particular epigenomics) across animals (in particular livestock). Its data modelv is also an extension of the INSDC model, also with recommended attributes and ontologies.\n\nICGCvi gathered the data from over 90 different cancer genome projects, as well as their heterogeneous analyses. The Data Coordination Center defined a set of data modelsvii, which is able to hold both anonymized metadata of the patients and samples, metadata of the experiments and analyses, as well as the results. It also established a federated cancer database, where the different partners are responsible for pushing their patients, samples, experiments and analysis metadata and data, having been translated and normalized to the ICGC data models. It became the seed for the more complete initiative of “Pan Cancer Analysis of Whole Genomes” (PCAWG)viii.\n\nIn addition, the Genomic Data Commons (GDC)ix is a research program of the National Cancer Institute (NCI) to provide the cancer research community with a unified data repository that enables data sharing across cancer genomic studies in support of precision medicine. Data and metadata are submitted to the GDC in standard data types and file formats through the GDC Data Submission Pipeline. The GDC hosts and distributes previously generated data from The Cancer Genome Atlas (TCGA), Therapeutically Applicable Research to Generate Effective Treatments (TARGET), and other programs. The GDC data model is based off the DAta Tags Suite (DATS)11, a general metadata model for biological results, which itself was designed to mirror the Journal Article Tag Suite (JATS)x, required for submission into PubMedxi to index publications.\n\nFinally, the ISA frameworkxii provides metadata standards for annotating experimental datasets, with detailed metadata configurations designed by expert groups for most common experiment types and domains, represented in ISA-TAB or ISA-JSON formats. The ISA data model is built around some core metadata categories: Investigation, Study, and Assay. In addition, the ISA framework provides tools for annotation, curation, or conversion of metadata, and also deployment tools that follow the requirements of public repositories, such as ArrayExpressxiii and the European Nucleotide Archive (ENA)xiv, as well as selected journals. Noteworthy, the ISA framework has recently been selected as one of the Recommended Interoperability Resourcesxv by ELIXIR.\n\nOther organizations are important as mechanisms to promote the adoption of standards by the international research community and include, for instance, the Global Alliance for Genomics and Health (GA4GH)xvi. This is an organization focused on the creation of policy frameworks and technical standards, which allow sharing of genomic and medical dataxvii in a responsiblexviii way. While much of the data types covered by GA4GH are not genomic tracks, some of the datasets can be displayed as genomic tracks, for example genotype data. Of relevance is also the refget APIxix, a GA4GH-approved standard that provides unambiguous access to reference sequences from unique checksum identifiers based on the sequence content itself.\n\nIn practice, standards are often defined and implemented by widely used tools, who set de facto standards on what is a valid input or not. We therefore list below some main consumers of track hub files.\n\nThe Track Hub Registryxx serves as a common entry point to register data collections into Ensembl12 and UCSC13 genome browsers. Its exchange format is a set of text files, collectively referred to as a track hubxxi. In effect, each genomic track is necessarily assigned the information required to display it. In particular, this includes its URL, the genome it maps to and free text descriptions. Optional attributes can then be attached to each track, in particular display settings, but also experimental metadata. Track metadata can be inserted into the track hub, either a) directly in the track hub file, as a list of key/value pairs, b) as an ancillary TSV file, or c) as an ancillary TagStorm filexxii.\n\nOn August 1st 2018, we surveyed the content of the Track Hub Registry. There were 4,294 track hubs spread across 10 species that all combined accounted for a total of 103,301 genomic tracks. Sampling through the BLUEPRINT track hub shows that each track contains on average 1.8GB of data. Because Ensembl have fully adopted track hubs and produce them automatically, most of the track hubs map to plant species (Arabidopsis thaliana, Zea mays, Oryza sativa Japonica Group, Glycine max, Sorghum bicolor, Triticum aestivum, Solanum lycopersicum, Brachypodium distachyon, Brassica napus), while only 3% map to human. However, the average human track hub has far more tracks (mean 218 tracks per hub) than the average plant track hub (mean 18 tracks per hub). A survey of metadata keys available on the track hubs suggests a great disparity in usage as provided in Table 1.\n\nThe Zenbu browserxxiii was initially developed to distribute the data produced by the FANTOM consortium. Data can be uploaded to it via its OSCTable formatxxiv, a general tab-delimited file format that subsumes the BED, GFF and SAM files used by genomic browsers. One distinction is that the format allows the writer to explicitly define data types and experiments (string identifiers). In addition, the file header allows the writer to optionally insert metadata describing the file or the experiment identifiers defined in the column names.\n\nThe Genomic HyperBrowser14 is a web-based framework aiming to provide a complete downstream solution for statistical analysis of track data, starting after data processing steps like ChIP-seq peak calling or variant calling has taken place. The implemented analyses are based on rigorous statistical fundamentals and methodologies, and include colocalization analyses, but also analyses making use of signal values and 3D DNA structure, as well as methods for clustering and visualisation.\n\nThe latest expansion, GSuite HyperBrowser1, constitutes a redesign of the analysis workflow with a focus on defining and managing collections of related tracks with associated metadata throughout all analysis steps. With the GSuite expansion, tools were created for searching and downloading data from established track repositories, such as Roadmap Epigenomics or ENCODE. This work laid bare a range of issues with the then current state of track metadata, starting a process that has led to the realisation of this implementation study.\n\nSeveral efforts have been carried out to collect and organise track data, presented for use as reference data for specific analysis methodologies, including EpiExplorer15, ColoWeb16, GenomeRunner17, LOLAweb18, and epiCOLOC19. Typically, the user provides a query track as input, which is then compared with the available reference datasets in order to generate relevant associations. Overall, such efforts are limited to organize tracks based on a few metadata fields, such as project, tissue type, assay type and assay target. Also, such track collections are often collected and organized prior to publication, but they are seldom updated afterwards. Similar efforts in the future would benefit significantly by the FAIRification of track metadata suggested here, as would other tools and services providing downstream analysis of track data, including machine learning methods20–22.\n\n\nMetadata recommendations\n\nBuilding upon prior art, we started by creating a data model around four key objects: studies, samples, experiments, and tracks (see Figure 1). The atomic data element is the Track, a genomic data file, generally in a binarized and indexed file format, such as BigBedxxv, BigWigxxvi or BCFxxvii, optimised for display in a genome browser. Each track is generated by an Experiment, whether physical or in silico. Physical experiments can be mapped for example to experiments in the European Genome-phenome Archive (EGA)23, although to our knowledge, in silico experiments do not have an authoritative identifier system. Physical experiments link out to Samples, as described in BioSamples24. Next, a set of experiments are contained within a study, which is akin to the EGA Study objects. Finally, one or more tracks are grouped into a Track Collection, which is the outer scope of the model and directly matches the existing track hub object. A Track Collection can also refer to an ad hoc collection of tracks, e.g., documenting the input data of published analyses.\n\nWe then defined the required and optional attributes for each object type (Table 2). The first consideration when defining a data model is to strike a compromise between the work imposed on the producer and the consumer of the metadata. The file must be clear and useful, yet not be such of a burden to compile that submitters would circumvent metadata entry. We therefore opted to only require attributes that appeared necessary for generic re-analysis of the data, while at the same time promoting FAIRness, by inclusion of resolvable references to relevant existing metadata records in external resources. To ensure practical usability, we generated a large test metadata record to test our proposal for ambiguities and difficulties, as described in more detail below. This testing phase clarified a need for an automated intermediate step between metadata curation and consumption, which in most cases simply meant to augment resolvable identifiers with human readable versions of the same. Adding this augmentation step simplified metadata entry, while simultaneously strengthened metadata search and extraction, in both automated and manual usage scenarios.\n\nOne major design consideration was allowing for multiplicity in the relations between these types of objects. For example, a sample can be associated with multiple experiments and an experiment with multiple track files (e.g., different analysis results), but, conversely, a track file can be linked with multiple samples (e.g., an aggregated statistic). Given these constraints, a hierarchical model was not possible, so we opted to define these objects independently, and then link them up by reference.\n\nThe schema was finally formalised as a JSON Schema, as this generic technology is widely used, and benefits from well-maintained parsers and validators in an array of programming languages. To a large extent, we re-used existing FAIR resources, such as ontologies or identification services that can be referenced with a CURIE and resolved via the Identifiers.org25 service, which maps a CURIE to the Internet locations where the referenced item is hosted.\n\n\nPrototype implementation\n\nHaving defined a theoretical standard, we tested its feasibility and usability by generating a test object that fits this specification. To ensure robust testing of our framework, we chose one of the largest existing track hubs, namely the BLUEPRINT track hub, formatted its metadata according to our proposed requirements, and then propagated it via representative services in the genomic track ecosystemxxviii:\n\nThe Track Hub Registryxxix allows data producers to register and share trackhub files. These files are then parsed and indexed by the Ensembl and UCSC genomic browsers and are thus available for searching on both of these widely used services. Currently, the main requirement for submission into the Track Hub Registry is to provide a correctly formatted track hub file that can be displayed on the genomic browser. Because its purpose is to facilitate the sharing and distribution of genomic analysis results, we do not plan to alter these requirements. Rather, datasets that conform to the metadata standard presented here would be highlighted and made easily available for transfer to downstream tools. To facilitate such transmission, the Track Hub Registry’s API now allows remote querying of its content.\n\nThe FAIRtracks validation serverxxx provides a RESTful API to allow FAIRtracks standard adopters to check whether the track metadata correctly adheres to the FAIRtracks JSON Schema. The server is based on standard JSON Schema validation technology, extended with additional Python modules that allow powerful local checks, such as validating ontology terms against specific ontology versions, or checking CURIEs against the registered ones at Identifiers.org. The extended validator also supports document-set restrictions, like unique constraints enforcement and foreignProperty checks. The JSON Schema validator extensions are being implemented also in Java.\n\nThe FAIRtracks augmentation servicexxxi is a RESTful API that takes as input a FAIRtracks-annotated JSON document containing the minimally required fields and automatically generates an extra set of “augmented” fields, containing human-readable ontology labels, ontology versions, and otherwise useful content for downstream users.\n\nTrackFindxxxii is a search and curation engine for FAIR genomic tracks. It supports crawling of the Track Hub Registry and other data portals to fetch track metadata. Crawled metadata can be accessed through hierarchical browsing or by search queries, both through a web-based user interface, and as a RESTful API. TrackFind supports advanced SQL-based search queries that can be easily built in the user interface, and the search results can be browsed and exported in JSON or GSuite formatxxxiii. The RESTful API allows downstream tools and scripts to easily integrate a TrackFind search, currently demonstrated by the GSuite HyperBrowser and EPICO. We plan to extract the curation functionality from TrackFind into a separate toolset, and extend it according to user needs.\n\nJSON-to-GSuitexxx is a RESTful service for converting FAIRtracks-annotated JSON documents into the GSuite formatxxxiv. This conversion is needed primarily to enable the TrackFind client in the GSuite HyperBrowser to output track metadata in the GSuite format, in order for the track collections that results from particular search queries to be consumable by existing manipulation and analysis tools in the framework.\n\nThe EPICO Data Analysis Portal is a generalization from the BLUEPRINT Data Analysis Portalxxxv. It is designed following the client-server paradigm, which also supports having different pluggable data backends at its REST API. The integration with the FAIRtracks ecosystemxxxvi is realised through a new ‘fairtracks’ backend, which translates queries and the data model to both the Track Hub API and to remote genomic tracks. Also, the REST API and the underlying EPICO data model has been generalized to deal with different reference genomic assemblies and organisms. The EPICO web frontend is also being updated, first to support dealing with different organisms and multiple reference genomic assemblies. Lastly, additional views are in preparation, in order to provide insightful views for the different kinds of genomic tracks, based on the various kinds of analyses and experiments to be undertaken.\n\nThe GSuite HyperBrowserxxxvii has been extended with a webtool that allows for querying FAIRtracks-annotated metadata available through TrackFind. This TrackFind client tool creates a GSuite file containing metadata for the resulting tracks, including remote URLs to the relevant track files. Existing tools in the framework can further download the track data to the server and prepare them for further analysis using the statistical analysis tools included with the GSuite HyperBrowser extension1. As a proof-of-concept, the BLUEPRINT metadata were queried and used in a demonstration analysisxxxviii of Multiple Sclerosis-associated DNA GWAS variants vs. DNAse I hypersensitivity sites from 58 BLUEPRINT normal cell type samples.\n\n\nDiscussion\n\nWe have presented a summary of existing metadata standards. We think there is an unmet need (Figure 2) for a standard for track metadata, together with a related infrastructure, aimed primarily at simplifying the day-to-day activities of researchers integrating genomic track data of varying types and from different sources, whether this is by manual web access, by ad hoc scripting, by the use of track analysis tools, or by the implementation of novel methodologies as new tools. Of great importance in such scenarios is how well a solution improves on the relatively poor state of accordance to the FAIR recommendations5 with current track metadata. The following discussion of our FAIRtracks draft standard and the related proof-of-concept infrastructure is thus organized according to the FAIR principles.\n\nF1. (Meta)data are assigned a globally unique and persistent identifier\n\nThere are few solutions for assigning globally unique identifiers to track files themselves. In many cases, only the raw sequence files are assigned identifiers, typically accession number to core data repositories, such as the Sequence Read Archive (SRA)26 or the European Genome-Phenome Archive (EGA)23. The ENCODE10 project represents a welcome exception, as they have assigned Identifiers.org-resolvable identifiers to each single track file with associated web-pages.\n\nTrack files typically contain condensed data returned from bioinformatics tools, which are run as part of defined workflows. As such, the track files are prone to contain biases due to specific parameter settings or workflow states, warranting the common advice to researchers to redo all analyses from the sequence files, with full control of the complete process. However, this might be impractical or unfeasible. Furthermore, track analysis is often exploratory in nature and a certain amount of error is often acceptable. The ability to uniquely identify specific track files is thus crucial for track analysis with positive consequences for reproducibility27.\n\nA globally accessible service to assign and register identifiers to single track files and collections of tracks is currently missing. We strongly recommend the implementation of a track registry that also preserves the full context surrounding the track files, with global identifiers to not only the track files, but also to the associated metadata. Our FAIRtracks draft standard could be advantageous for use as a basis for such a registry of track metadata. For now, we are leveraging the widespread adoption of the document identifier (DOI) by requiring a FAIRtracks document to be published and identified with a DOI. We require the publisher to support DOI versioning and also the possibility of reserving a DOI prior to publication (to include the DOI in the published file itself). We currently recommend using Zenodoxxxix as the publishing platform, as the service supports both features, but other platforms are also possible as long as both DOI versioning and reservation are possible. FAIRtracks is easily extendable to support other global identifier types.\n\nTo improve the provenance of analyses making use of genomic track data, e.g., as part of a publication, it would be useful to also be able to uniquely identify specific ad hoc collections of tracks. One way to support this would be to only allow a single track collection in a FAIRtracks document, and then refer to the DOI. There is, however, an issue with granularity, as the number of published datasets in that case would be impractically high, as no publishing platform supports identifiers at a sub-dataset level in combination with versioning and pre-reservation of DOIs. Creating a separate service to assign and register identifiers at the track collection level seems a better solution (see A2 below).\n\nF2. Data are described with rich metadata\n\nThis FAIR principle (and also R1) concerns the importance of providing generous metadata, and to not limit their availability due to assumptions about the users. This principle seems to stand in direct opposition to our goal of proposing a minimal standard for track metadata, based upon, precisely, our ideas of usability. However, the FAIR principles relate to the metadata content, and not its structure. FAIRtracks explicitly allows for any metadata property to be added by document creators. Indeed, the schema is designed so that existing repositories with curated track metadata can be converted in batch to follow FAIRtracks, retaining all the non-conforming metadata fields. Rather than being a schema for metadata completeness, FAIRtracks also advocates the inclusion of machine-resolvable identifiers that refer to external metadata records deployed in external community-standard repositories, such as BioSamples24.\n\nF3. Metadata clearly and explicitly include the identifier of the data they describe\n\nAs a global track repository currently does not exist, we were not able to require the inclusion of global identifiers to track data files. FAIRtracks does, however, require the inclusion of URLs to track files, but this is without any guarantees of persistence or uniqueness. Even if a track file does not have a direct identifier attached, one can often instead include an identifier to a parent record, e.g., to an experiment or a study. As FAIRtracks requires a global identifier for the metadata document itself (using DOI), it should be possible to uniquely identify a track file from a joint identifier containing the DOI of a FAIRtracks document and the local identifier for a track file within that document.\n\nOne of the main goals of this study has been to make it easier for researchers to make use of historical track sources. Unfortunately, many existing track sources contain little metadata and seldom identifiers referring to external records of, e.g., samples or studies, and also no domain standard for which external repositories to refer to. We thus concluded that we at least for now would recommend, but not require, the inclusion of global identifiers to external records for the main object types.\n\nF4. (Meta)data are registered or indexed in a searchable resource\n\nIn parallel to the specification of the FAIRtracks draft standard, we have implemented a central search service named TrackFind. Our experience from the development and usage of track analysis software has clearly shown that the most useful form of track search is a formal search with a predefined set of values for each attribute, in contrast to a free-text search interface which is a typical choice for integrative services. The usefulness of such categorical metadata can be witnessed in the design of most track data portals. For instance, in the IHEC Data Portalxl, the datasets are even visualized as a matrix defined by categories of epigenomes (e.g., cell or tissue types) in one axis, and assay targets (e.g., transcription factors or histone modifications) in the other. The main challenge in order to make such a search service work across categorical metadata from a range of sources is in harmonizing the metadata.\n\nWe have attempted to harmonize metadata at different levels: 1. We have selected and named a set of core attributes. 2. We have limited the possible values for these attributes to those contained in certain ontologies or limited vocabularies. 3. We have tried to balance the various constraints posed on the metadata structure from, e.g., specific types of track data, experimental techniques, biospecimen types, or ontology richness, with the need for uniform metadata attributes and categories across heterogeneous track sources and data types. With FAIRtracks, we believe we have managed to balance the various constraints in a manner that makes the schema uniquely suitable for a unified formal search service. With TrackFind, we provide a rich web interface that combines hierarchical browsing of attributes and values with a solution for piecing together advanced queries. In addition, we provide a REST API for use by other software tools or for ad hoc scripting. Also, the TrackFind client tool in the GSuite HyperBrowser provides a helpful step-by-step search interface, where each selection limits the values that can be selected in the next step, in concordance with the metadata contents. In conclusion, we believe TrackFind and the FAIRtracks draft standard together will be able to greatly improve the findability of track files.\n\nA1. (Meta)data are retrievable by their identifier using a standardised communications protocol\n\nIn our prototype implementation, indexed metadata are accessible through simple REST-based APIs that use the HTTP protocol, either from the Track Hub Registryxli or from TrackFindxlii. The track data as such are available by FTP or HTTP, as hosted by the data provider. In some cases, e.g., for human variation data, access to the track data will be restricted, in which case we provide specific metadata fields for linking to the data usage policy and access control procedures. We also support the possibility to include identifiers to the raw files used to create the track, resolvable to external repositories. We advise to include such raw file references, as only referring to a higher-level dataset record will make it difficult to locate the exact files to use for researchers seeking to redo the upstream pipeline. However, in our test case of track files from the BLUEPRINT project, we were not (with reasonable effort) able to extract the EGA identifiers to the raw data files from the metadata available to us. Conversely, the ENCODE project provides access to raw files in an exemplary manner by providing multiple levels of references to original files, together with metadata about the pipeline that was used for each step.\n\nA2. Metadata are accessible, even when the data are no longer available\n\nMetadata persistence is a particularly important issue for genomic track files, as it is a type of data that is especially prone to becoming unavailable due to a technicality in how most genome browsers operate, in that they allow remote hosting of track files in file formats that support this, such as BigBed and BigWig28. However, for this to work, the hosting web server needs to be configured with the ability for users to fetch only specific subparts of the files. Some common repositories for life science data do not support this, leaving the data providers with a need to host the files themselves, often achieved using temporary web hosting services. Thus, track data files often become unavailable after some years.\n\nAs a minimum, track metadata should persist. Even though Track Hub Registry (THR) and TrackFind could technically be able to fill such a role, we choose to depend on existing persistent repositories using DOI identifiers (e.g., Zenodo, see F1), as the operational model of THR allows submitters to delete their submissions, while the architecture of TrackFind is primarily designed around its search functionality. Zenodo provides storage connected to the CERN project infrastructure, to be maintained for at least 20 yearsxliii.\n\nOne solution for providing persistent identifiers at the level of track files and track collections would be to implement a light-weight track registry service that builds on existing infrastructure. Such a service could: 1. Accept a DOI to a FAIRtracks-formatted document. 2. Generate and register globally unique identifiers for the contained track files and track collections. 3. Add these identifiers to the document. 4. Store the document in an existing persistent repository. 5. Allow fine-grained access to the metadata for each track or track collection. 6. Provide references (through at least Identifiers.org) to such fine-grained access to the document, as well as to the relevant records in TrackFind and elsewhere. 7. Possibly host web pages with track metadata using Bioschemasxliv mark-up for improved findability through standard web search engines. 8. Possibly support persistence of track data itself (e.g., through BioStudiesxlv), given the availability of the required storage space.\n\nI1. (Meta)data use a formal, accessible, shared, and broadly applicable language for knowledge representation\n\nWe tried to define the FAIRtracks model in a way that objects could be easily mapped to objects in other relevant metadata models (Table 3). Thus, the FAIRtracks objects \"Experiment\", \"Study\", and \"Sample\" refer one-to-one to the INSDC counterparts with the same name and relationshipsxlvi, with the exception that a FAIRtracks experiment might also refer to an in silico analysis, like a ChIP-seq peak calling run, in INSDC covered by the \"Analysis\" object. Compared to the ISA Abstract Modelxlvii, the FAIRtracks \"Study\" and \"Sample\" objects match identically named ISA objects, while the FAIRtracks \"Experiment\" object corresponds to either ISA \"Assay\" or \"Process\" objects. The addition of the \"aggregated_from\" attribute to the FAIRtracks \"Experiment\", allows downstream bioinformatics analyses to be traced stepwise back to the original laboratory experiment, in a more expressive way compared to the INSDC model, as well as in a simpler way compared to the ISA Abstract Model. The FAIRtracks \"Track\" object covers a single track file, which in INSDC is also confusingly covered by the \"Analysis\" object, and in the ISA Abstract Model by the \"Data\" object. We have also added an additional \"raw_file_ids\" attribute to the track record in order to allow matching track files with the exact original data files. Lastly, the FAIRtracks \"Track collection\" object is similar to the SRA \"Submission\", EGA \"Dataset\", Track Hub Registry \"Track Hub\", and ISA Abstract Model \"Investigation\" object. In addition, a track collection might also be used to describe ad hoc collections of track files extracted from different repository submissions, e.g., to uniquely refer to a set of track files analysed in a research paper, thus providing a novel way to improve reproducibility of research findings. Used in this way, a FAIRtracks \"Track collection\" directly corresponds to the contents of the GSuite metadata file format, previously developed in context of the GSuite HyperBrowser1, and we have thus also implemented tools to convert between the two formats.\n\nWe have chosen JSON as the formal exchange format for track metadata. In addition to being the de facto standard for web APIs, JSON metadata can also be formalized at a meta-meta level using JSON Schemaxlviii. We thus developed the FAIRtracks draft standard as a set of JSON Schemas together with a validation service. Furthermore, we found the need to extend the FAIRtracks schemas with more property types than was allowed by the version of JSON Schema it follows (draft 7). Specifically, we extended validation to relationships between data (identifier existence and uniqueness within a FAIRtracks document, a type of validation that is outside the scope of JSON Schemaxlix), as well as the validation of ontology terms and Identifiers.org CURIEs. Our validation service is a generalization of software initially implemented in the context of the ELIXIR OpenEBench projectl, illustrating the general applicability of a possible standardized extended JSON schema and validation service for life sciences, as also investigated by the ELIXIR Implementation Study on Data Validationli.\n\nI2. (Meta)data use vocabularies that follow FAIR principles\n\nFAIRtracks is following principle I2 by requiring that almost all text-based properties follow certain ontologies. For each such property, we have specified one or more supported ontologies, in most cases together with a set of ancestor terms. Each ontology term property consists of a pair of properties, one containing the term ID (as a URL), while the other contains the associated human readable label for the term. All of these relations are thoroughly validated by our FAIRtracks validation service.\n\nWe have not conducted thorough research of the FAIRness of the ontologies themselves, as this has been better handled by more dedicated studieslii. We have, however, faced challenges in three areas: findability, provenance and cross-ontology references.\n\nFirst, it is difficult to evaluate and compare the quality of ontologies for a particular domain. However, most relevant ontologies are helpfully registered in the Ontology Lookup Serviceliii (OLS) and the NCBO BioPortalliv, which both provide support for ontology discovery based on a limited set of metadata fields. The OBO Foundrylv provides manually curated lists of OBO ontologies. FAIRsharinglvi annotates ontologies with richer metadata and provides a better experience for discovering ontologies. The AgroPortal29 provides an expressive search interface built on a general model created by merging standards for metadata on ontologies, but the portal only supports ontologies in agronomy and related domains. In practice, we chose ontologies manually by getting an impression by searching for key terms, using e.g., OLS or Zoomalvii, followed by unstructured browsing of the resulting ontologies. Such a process was obviously highly subjective, in addition to having limited usefulness if one does not possess expert knowledge in the relevant domains. We would have been greatly helped in this if objective ontology metrics were readily available to guide us in our choices, e.g., based on related databases, standards, and policies as annotated in FAIRsharing.\n\nSecondly, the FAIR principle R1.2 recommends provenance of both data and metadata, however we are unaware of clear recommendations and solutions for capturing the exact version of an ontology that is used when annotating data. Changes in ontology structure and content may create problems for automatic validation or search. Initially, we figured that a specific version of our FAIRtracks draft standard should require ontology terms according to specific versions of each ontology, ensuring consistent validation of ontology terms. However, this would require us to release an update of FAIRtracks for every new release of a supported ontology, and it would also complicate the implementation of the validator. In the end, we opted to not require specific versions of the ontologies and instead validate against the latest version of each. However, we require that the ontology versions are captured for each metadata document. With this solution, ontology changes might break validation of previously validated metadata records, but since provenance is available, one should at least be able to resolve any issues.\n\nThirdly, managing cross-ontology references is a fundamental challenge that was not fully explored within the scope of this study. We therefore in most cases chose to support only a single ontology for each term. Without this limitation, the list of allowed values for a field would typically end up containing variations of the same terms, making downstream use unnecessarily cumbersome, e.g., when selecting filters for a query. On the other hand, limiting to only one ontology for each field places a burden on the metadata providers and curators, as one would probably not be able to find an ontology scheme that suits the particular use case or domain for every track collection. FAIRtracks can potentially allow non-redundant merging of several domain-focused ontologies by making heavy use of ancestor term restrictions. Regardless, there is a clear need for powerful tools to translate terms across ontologies.\n\nFor our BLUEPRINT test case, we made good use of the Ontology Xref Service (OXO)lviii to transform existing ontology terms in order to comply with our FAIRtracks draft standard. OXO is still under development and the conversion process required some manual oversight, for example to select between alternative mappings. We note that some projects such as GWAS Cataloglix already automated the process of batch transformation and mapping of structured metadata, tailored to their use cases (pers. comm. H. Parkinson).\n\nThe FAIRtracks draft standard includes two novel fields in the Track object that we believe will prove to be very useful for downstream users, but that we were unable to properly populate using existing ontologies. We thus decided, for now, to provide limited vocabularies for them in the JSON schema itself. The aim of both fields is to provide a simple theoretical framework to categorize track files according to their potential usage. Track data, by definition, is formed downstream of some data condensation process, typically a pipeline of software tools that simplifies a set of raw sequence reads of varying quality into a condensed track file with relatively high-confidence data. Several existing ontologies contain terms describing the experimental technique, e.g., \"ChIP-seq assay\"lx, as well as the pipelines, e.g., \"Peak calling\"lxi, but there are very few terms available to describe the results of those pipelines, that is, specific types of condensed data represented as track files, e.g., \"Broad peaks\". In contrast to file formats, e.g., \"BigBed\"lxii, our first novel field \"type_of_condensed_data\" describes the condensed data itself, and thus its interpretation, rather than its representation. The second field, \"genomic_track_type\", builds upon a previous study7 delineating tracks according to their basic geometric properties when viewed as mathematical objects along a one-dimensional line (i.e. the coordinate system defined by the reference genome assembly). Our initial vocabularieslxiii are not meant to be comprehensive lists, but rather starting points of a process leading to the inclusion of such terms into a relevant ontology, possibly the EDAM ontologylxiv where such data-describing terms might fit well.\n\nAs the FAIRtracks draft standard is developed primarily as a proof-of-concept and a starting point for a common metadata standard, our initial choices of supported ontologies are suggestions open for debate, mostly based upon our opinions and that of early adopters. However, as the standard matures, we expect the choices to converge towards a set of broadly supported, and FAIR, ontologies.\n\nI3. (Meta)data include qualified references to other (meta)data\n\nRather than aiming for metadata completeness, FAIRtracks aims to improve track data and metadata availability and reuse by bridging the current gap between track data repositories on one hand, and analysis scripts, tools and frameworks on the other. As discussed in detail above (F2, F3), FAIRtracks thus supports and recommends the inclusion of global identifiers to external records containing more detailed metadata. We require that such global identifiers are represented in CURIE form to be resolvable through the Identifiers.orglxv service, one of the ELIXIR Recommended Interoperability Resourceslxvi. Due to a recent technical collaboration25, all supported CURIEs should also be resolvable through the US-based N2T.net servicelxvii, but this has not been tested. A mapping service in the opposite direction to Identifiers.org, i.e., from existing URIs to the corresponding CURIEs, would be highly useful for adapting existing metadata to the FAIRtracks draft standard.\n\nHistorically, lacking or inaccurate identification of the genome assembly version of track files has been a source of great confusion6, and can serve as an example. Even when assembly identifiers such as \"hg19\" and \"GRCh37\" are included in the track metadata, they lack the required context for interpretation, causing researchers to have to delve into the intricacies surrounding naming conventions for human genome assemblies. Full URIs, such as \"https://www.ncbi.nlm.nih.gov/assembly/GCF_000001405.13\", refer uniquely to unambiguous records, and thus improve on simple string and/or number combinations. However, using URIs require the records to persistently be available at a particular location. The CURIE counterpart \"insdc.gca:GCF_000001405.13\" is location-independent, and should resolvelxviii into both the above URI and a URI for a record stored within the EMBL/EBI infrastructure.\n\nAs a side note, using such a CURIE does not solve the inconsistencies on chromosome naming conventions for certain species (e.g., human and mouse). Even though the Genome Reference Consortium (GRC) standard denotes chromosomes using simple numbers and capital letters (e.g., 1, 2, X, ...), the historical convention of the UCSC Genome Browser (e.g., chr1, chr2, chrX, ...) is still the de facto standard for most track files. In FAIRtracks, we have tried to solve this issue by requiring an additional field \"annotation_name\" containing either the standard name of the genome assembly (e.g., \"GRCh37\") or one of the registered synonyms (e.g., \"hg19\"), according to the chromosome naming scheme used in the track file. Ongoing GA4GH-supported community efforts to standardize unique identifiers for collections of sequences should improve the situation, and will likely be adopted in a later version of FAIRtracks.\n\nA potential limitation to our extensive use of resolvable identifiers is the added requirement that all identifiers are openly resolvable from a web endpoint. In the case of the BLUEPRINT test metadata, we discovered for instance that the EGAX experiment identifiers were not resolvable (only EGA dataset and study types were supportedlxix). Adding such support would require the implementation of a dedicated API endpoint, a decision that in many cases is not up to the metadata provider. The current consequence is that our test BLUEPRINT metadata set does not validate correctly. It remains to be seen whether increased usage of CURIEs over time will decrease the occurrence and impact of such problems.\n\nR1. Meta(data) are richly described with a plurality of accurate and relevant attributes\n\nFAIR principle R1 overlaps somewhat with principle F2, but while F2 focuses on discovery of the dataset, R1 focuses on the user's ability to decide whether the data is useful in their particular research scenario. We believe our minimal set of required metadata fields, extracted from existing standards and selected based on years of experience in track file analysis, provides a breadth of generally relevant fields for most typical usage scenarios. Also, FAIRtracks enforces quite stringent limitations on the contents of the fields in order to provide sufficient accuracy. Furthermore, richer metadata can be added to external resources referred to by the FAIRtracks records, or such metadata can be added directly to the FAIRtracks records themselves.\n\nWe realized during the implementation process that two seemingly conflicting views of the purpose of our draft standard were held within our project group. These two views can be summarized as follows:\n\n#1. Facilitating the minimal set of fields that a submitter of a track collection would need to fill out in order to get the metadata approved as FAIR\n\n#2. Facilitating the minimal structured metadata that is directly useful for downstream usage\n\nAs an example, consider the case of ontology terms. Metadata submitters should only need to provide the correct identifier for each term. However, those identifiers are incomprehensible for downstream human users. If the standard were to follow view #1, all downstream tools would thus need to implement ontology lookup functionality, making the process unnecessarily cumbersome for researchers, especially in simple scripts. However, if the standard were to fully follow idea #2, the burden of ontology lookup would be placed on the data providers. Similar conflicts were present for other issues. Fortunately, we discovered a technique that would help us adequately meet the requirements of both usability and ease of metadata entry: We implemented an augmentation servicelxx,lxxi that, based on the minimal fields required by the FAIRtracks JSON schema (supporting view #1), automatically generates an extra set of \"augmented\" fields, containing human-readable or otherwise useful content for downstream users, removing the need for ontology lookup and similar (supporting view #2).\n\nWe have also found that augmentation can be very useful for integrating heterogeneous datasets, e.g., for multi-omics analysis. Rich and accurate metadata fields are often highly specific to particularities of a type of datasets, e.g., related to sampling process or experimental technique. However, having to gather and integrate metadata values from such specialized fields is impractical if one is integrating a number of different dataset types. Consider, e.g., two samples: one is derived from an established cell culture, while the other was created by isolating a particular cell type from a biological sample. In the first case, a main categorization of the sample could be represented by a \"cell line\" field, which would only accept values according to an ontology of cell lines, e.g., \"H1-hESC\". In the other case a controlled \"cell type\" field could be used for the categorization, e.g., as \"B cell, CD19 positive\". However, a researcher might want to merge such fields into one for a broad scan of available data. Or similarly, a researcher might want to combine ChIP-set tracks of transcription factor binding alongside histone modification experiments, or even GWAS variants for different phenotypes. The 2-standards-in-1 design of FAIRtracks allows the support for both stringent validation of clearly defined fields to ease metadata entry, while at the same time providing merged fields with harmonized content. The FAIRtracks draft standard includes two such summary fields populated by the augmentation service. First, in the \"Sample\" object, the \"sample_type → summary\" field summarizes the other child fields of \"sample_type\", i.e. \"abnormal_cell_type\", \"cell_line\", \"cell_type\", and \"organism_part\", according to rules determined by the value in the field \"biospecimen_class\". Second, in the \"Experiment\" object, the field \"target → summary\" is an augmented field containing the summary of the other child fields of \"target\", i.e. \"gene_id\", \"gene_product_type\", \"macromolecular_structure\", \"phenotype\", \"sequence_feature\", and \"target_details\", according to rules determined by the value of the field \"technique\". In the case of, e.g., ChIP-seq transcription factor (TF) experiments, the HUGO Gene Nomenclature Committee (HGNC) identifier for the TF should be entered in the \"gene_id\" field, with any post-translational modifications in the \"target_details\" field. Such rules are enforced by heavy use of JSON Schema validation structures.\n\nIt can be argued that it is risky to define a general standard to include metadata requirements based on certain particularities of specific experimental types and sample types, as the maintenance of up-to-date and domain-relevant logic might become difficult. However, we believe the advantages of such limited logic significantly outweighs the extra burden. We envision to gradually extend and improve the logic and structures with a group of early adopters based on real-world metadata.\n\nR1.1. (Meta)data are released with a clear and accessible data usage license\n\nThe FAIRtracks draft standard annotates track data files with a combination of the machine-readable fields using the GA4GH-approved Data Use Ontologylxxii, as well as a URL field linking to human-readable data use license/policy documents.\n\nR1.2. (Meta)data are associated with detailed provenance\n\nIt has been paramount to the design of the FAIRtracks draft standard, as well as to the supporting infrastructure, to allow tracing the provenance of track data and metadata in a transparent manner. This is handled at many levels:\n\n1. FAIRtracks is an intermediate metadata standard designed to include references to external metadata records, including the original records from which the FAIRtracks metadata has been derived. Those records will often contain additional provenance information.\n\n2. As detailed under I1 above, FAIRtracks supports a mechanism for tracing the data stepwise back through various bioinformatics analyses and laboratory experiments, by using the field \"aggregated_from\" in the \"Experiment\" object. One can attach external records to every step, if available.\n\n3. A separate mechanism, the field \"raw_file_ids\" in the \"Track\" object, can be used to at least refer to the original data files, even if a stepwise history is not available.\n\n4. The \"Experiment\" object contains the free-text fields \"lab_protocol_description\" and \"compute_protocol_description\" to record information about the experiment setup, either directly or as a URI to an external record. Optimally, such provenance information should be stored in a structured manner, according to community standards for provenance information, such as Provenance Notation (PROV-N)lxxiii or CWLProv30. Records containing provenance information could be attached to globally unique and persistent identifiers and made available (e.g., as RO-Cratelxxiv)31, possibly also in a searchable registry, such as the ongoing development of WorkflowHublxxv. As research and implementation of experiment provenance metadata is out of scope of the current project, we settled for free-text fields.\n\n5. In the top-level metadata relating to a FAIRtracks document itself, we include fields for version information and date, as well as \"derived_from\" and “augmented_from” fields that can be used to trace the evolution of the metadata itself. Such provenance tracing requires globally unique and persistent identifiers for track metadata content, as we argue for in section F1 above.\n\nR1.3. (Meta)data meet domain-relevant community standards\n\nCurrently, efforts of FAIRifying genomic track data are mostly restricted to a few high-volume providers, with related metadata guidelines. Those few community standards that exist are either defined for specific domains, e.g., human epigenomics (IHEC) or animal genomes (FAANG), or in relation to specific tools or services, e.g., the TrackHub format specification or the Track Hub Registry requirements. None of the few community standards have been designed for general use and in explicitly in accordance with the FAIR principles. We have designed the FAIRtracks draft standard to combine broadness of metadata content with enough stringency to be trusted by downstream users and tools, based on years of experience as consumers of genomic tracks. We thus aim for the FAIRtracks draft standard to evolve into such a community standard, helped by the establishment of useful services that facilitate FAIR sharing of genomic track files, not least through the TrackFind web server and search API. We aim to approach core providers of data and tools to build an initial catalogue of content and integrations. As soon as a sizable amount of track metadata is available, we expect the momentum to integrate with TrackFind to increase, propelling further metadata FAIRification. For further adoption from data providers, we plan to provide automatic certification by flagging Track Hub Registry submissions that is verified to contain FAIR metadata, and we recommend the implementation of a track metadata registry. Such a registry should require submissions according to a FAIR community standard, possibly evolved from the FAIRtracks draft standard. We also intend to make FAIRtracks-related contributions to other FAIR sharing efforts, such as to the FAIRplus Cookbook, which is a resource that collates FAIRification recipes primarily in connection to the Innovative Medicines Initiativelxxvi, but also with an aim to reach as wide an audience as possible32.\n\nFurthermore, to simplify metadata transformation and curation, we envision taking part in community efforts to implement services for computer-assisted FAIRification of metadata, including mechanisms for curation and transformation of features, both on a per-record basis and in batch. It would be imperative for such services to provide advanced support for curating ontology terms, including guided ontology matching, support for generating CURIE identifiers, and integration with the FAIR metadata validation service.\n\nFor a community standard to evolve, the consolidation of a relevant community needs first to take place. The current project group, though relatively small, has included members with solid experience from tools development (GSuite HyperBrowser, EPICO); from track data and metadata production (BLUEPRINT, FANTOM); from consortia-based management of track repositories and related standards (IHEC, ICGC); as well as from the registration and dissemination of user-submitted track collections (The Track Hub Registry). An obvious weakness of our approach is the uncertain outlook on community acceptance of the FAIRtracks draft standard. Typically, standards are created either as part of early, domain-specifying implementations, or by larger field-dominating groups. However, standards also sometimes arise if they fill specific gaps of general importance, and it is our hope that a broader community will arise downstream of our efforts. The current study represents the initial step in this process.\n\n\nConclusion\n\nWe believe that with the proposed FAIRtracks draft standard and accompanying service infrastructure, we have taken a useful step towards realizing the underexploited value of genomic track datasets. However, whether this effort will realize the full potential will ultimately be decided by the scientific community, to the extent that the standard will be adopted and implemented.\n\n\nData availability\n\nNo data is associated with this article.\n\n\nSoftware availability\n\nFAIRtracks standard (including FAIRified BLUEPRINT metadata): https://github.com/fairtracks/fairtracks_standard (License: CC-BY 4.0)\n\nTrack Hub Registry: https://github.com/Ensembl/trackhub-registry (License: Apache 2.0)\n\nFAIRtracks validator (License: LGPL 2.1) and FAIRtracks validator server (License: AGPL 3): https://github.com/fairtracks/fairtracks_validator\n\nFAIRtracks augment service: https://github.com/fairtracks/fairtracks_augment (License: Apache 2.0)\n\nFAIRtracks format conversion service: https://github.com/fairtracks/fairtracks_json_to_gsuite (License: MIT)\n\nTrackFind: https://github.com/elixir-no-nels/trackfind (License: MIT)", "appendix": "Acknowledgements\n\nWe thank Abdulrahman Azab and Geir Kjetil Sandve for contribution to the track data import tool in GSuite HyperBrowser, which inspired this work.\n\n\nFootnotes\n\ni https://trace.ncbi.nlm.nih.gov/Traces/sra/sra.cgi?view=xml_schemas\n\nii https://ena-docs.readthedocs.io/en/latest/meta_01.html\n\niii https://github.com/IHEC/ihec-metadata/blob/master/specs/Ihec_metadata_specification.md\n\niv https://www.animalgenome.org/community/FAANG/\n\nv https://github.com/FAANG/faang-metadata/blob/master/docs/faang_metadata_overview.md\n\nvi https://icgc.org/\n\nvii http://docs.icgc.org/dictionary/viewer/\n\nviii https://dcc.icgc.org/pcawg\n\nix https://gdc.cancer.gov/\n\nx https://jats.nlm.nih.gov\n\nxi https://www.ncbi.nlm.nih.gov/pubmed\n\nxii https://isa-tools.org\n\nxiii https://www.ebi.ac.uk/arrayexpress/\n\nxiv https://www.ebi.ac.uk/ena\n\nxv https://elixir-europe.org/platforms/interoperability/rirs\n\nxvi https://www.ga4gh.org/\n\nxvii https://www.ga4gh.org/ga4ghtoolkit/genomicdatatoolkit/\n\nxviii https://www.ga4gh.org/ga4ghtoolkit/datasecuritytoolkit/\n\nxix https://samtools.github.io/hts-specs/refget.html\n\nxx https://trackhubregistry.org/\n\nxxi https://genome.ucsc.edu/goldenpath/help/hgTrackHubHelp.html\n\nxxii https://f1000research.com/posters/6-479\n\nxxiii http://fantom.gsc.riken.jp/zenbu/\n\nxxiv https://zenbu-wiki.gsc.riken.jp/zenbu/wiki/index.php/OSCtable\n\nxxv https://genome.ucsc.edu/goldenPath/help/bigBed.html\n\nxxvi https://genome.ucsc.edu/goldenPath/help/bigWig.html\n\nxxvii https://github.com/samtools/hts-specs/blob/master/BCFv2_qref.pdf\n\nxxviii https://fairtracks.github.io/\n\nxxix https://trackhubregistry.org/\n\nxxx http://fairtracks.bsc.es/api/\n\nxxxi https://fairtracks.elixir.no/api/\n\nxxxii https://trackfind.elixir.no/\n\nxxxiii http://www.gtrack.no/\n\nxxxiv https://github.com/gsuite/gsuite\n\nxxxv http://blueprint-data.bsc.es/\n\nxxxvi http://fairtracks.bsc.es/#epico\n\nxxxvii https://hyperbrowser.uio.no\n\nxxxviii https://hyperbrowser.uio.no/trackfind_test/u/hb-superuser/h/fairtracks\n\nxxxix https://zenodo.org/\n\nxl https://epigenomesportal.ca/ihec/grid.html\n\nxli https://www.trackhubregistry.org/docs/apis\n\nxlii https://trackfind.elixir.no/api/\n\nxliii https://help.zenodo.org/\n\nxliv https://bioschemas.org/\n\nxlv https://www.ebi.ac.uk/biostudies/\n\nxlvi https://ena-docs.readthedocs.io/en/latest/submit/general-guide/metadata.html\n\nxlvii https://isa-specs.readthedocs.io/en/latest/isamodel.html\n\nxlviii https://json-schema.org/\n\nxlix https://github.com/json-schema-org/json-schema-spec/wiki/Scope-of-JSON-Schema-Validation\n\nl https://openebench.bsc.es/\n\nli https://elixir-europe.org/about-us/commissioned-services/data-validation-2018\n\nlii https://github.com/sifrproject/MOD-Ontology\n\nliii https://www.ebi.ac.uk/ols\n\nliv https://bioportal.bioontology.org/\n\nlv http://www.obofoundry.org/\n\nlvi https://fairsharing.org/\n\nlvii https://www.ebi.ac.uk/spot/zooma\n\nlviii https://www.ebi.ac.uk/spot/oxo\n\nlix https://www.ebi.ac.uk/gwas/\n\nlx http://purl.obolibrary.org/obo/OBI_0000716\n\nlxi http://edamontology.org/operation_3222\n\nlxii http://edamontology.org/format_3004\n\nlxiii https://github.com/fairtracks/fairtracks_standard/blob/master/docs/fairtracks_track.md\n\nlxiv http://edamontology.org/\n\nlxv https://elixir-europe.org/platforms/interoperability\n\nlxvi https://elixir-europe.org/platforms/interoperability\n\nlxvii http://n2t.net/\n\nlxviii Personal comm.\n\nlxix https://registry.identifiers.org/registry?query=ega\n\nlxx https://fairtracks.elixir.no/api/\n\nlxxi https://github.com/fairtracks/fairtracks_augment\n\nlxxii https://github.com/EBISPOT/DUO\n\nlxxiii https://www.w3.org/TR/prov-n/\n\nlxxiv https://researchobject.github.io/ro-crate/\n\nlxxv https://workflowhub.eu/\n\nlxxvi http://www.imi.europa.eu/\n\n\nReferences\n\nSimovski B, Vodák D, Gundersen S, et al.: GSuite HyperBrowser: integrative analysis of dataset collections across the genome and epigenome. 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Reference Source" }	[ { "id": "82701", "date": "21 May 2021", "name": "Max Haeussler", "expertise": [ "Reviewer Expertise genome browsers", "genome annotations" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article proposes a standard for adding metadata to track hubs. The \"track hub\" file format was defined by the UCSC Genome Browser group to be able to display genome annotations from static files, without having to setup a server like DAS. It was not meant to be a general data exchange format, but nevertheless includes various ways to add metadata to the tracks. But the metadata key names are not defined and there are no required keys. This makes it hard to find annotation tracks, as in practice, most track hubs do not use the metadata at all, so all one is left with are the short and long human-readable labels. This makes finding and automated analysis of tracks (e.g. Chip-seq, variants, CNVs) harder than necessary.\nTo solve this problem, the article proposes a system where a JSON file is added to the track hub with data on study/sample/experiment and tracks and a given set of recommended key/value items for each track specified via ontologies.\nThe proposed solution sounds reasonable. If adopted by everyone, it would solve make track hubs adhere to the FAIR principles much better than at the moment. Similar to the authors themselves, I have doubts that this will be adopted quickly, but the paper is still sound.\nThe paper is well written as it is, I have no minor comments, just a few general ones:\n0) You say that the format must be \"clear and useful\". (Does this mean that simplicity was not a design goal?) I think the article would be easier to understand if you show a simple JSON example. The data format is explained, but examples go a long way. I know that you link an example on your Github page, but in an article where you discuss a new data format, an example right in the article of an example allows the reader to check that the format is indeed clear and useful.\n\n1) You note that the UCSC track hub specification already includes three ways to define metadata. You note that hardly any track hub uses the existing very minimal metadata system. Do you think that yet another metadata format will be used more than the current one? If yes, why? Do you have suggestions what be done by genome browsers, journal editors or databases to encourage hub developers to add more metadata, e.g. when ranking search results, showing track hubs, or when requiring authors to provide track hubs, like NAR does? These could fit very well in the discussion at the end.\n2) The existing metadata formats are either very simple (inline or tsv) or very compact (tagStorm). tagStorm and inline are human-readable text files, they can be typed easily in a text editor and they support, for example, comments. JSON does not support comments and is not as easy to type by humans. Do you think that is a disadvantage of JSON?\n3) \"The Track Hub Registry is a global centralised collection of public track hubs\" - the EBI's track hub registry is copying the UCSC public hubs list, which preceded it. The UCSC public hubs list is a curated list of track hubs submitted to UCSC and manually quality-checked. The Track Hub Registry adds to this list thousands of track hubs auto-generated from ENA submissions. In addition, some track hubs may have been submitted by users through the track hub registry API, but I see no way to find out which ones were added in which way. I think you could mention the UCSC public hubs list as a global, centralized and curated collection of public track hubs with certain quality standards. \"The Track Hub Registryxxix allows data producers to register and share trackhub files. These files are then parsed and indexed by the Ensembl and UCSC genomic browsers\" - There is a confusion in the article between the track hub registry and the UCSC Genome Browser. The Genome Browser does not \"index\" the track hub registry. By clicking the link on the registry, you open this hub. The UCSC Genome Browser indexes its own public hubs list for searching, so when you search for tracks, the Genome Browser will bring up tracks from the public hubs list, but not from the registry.\n4) You structure your data model around the usual (study, sample, experiments) entities, similar to the SRA. What happens if a dataset has a hierarchical structure within the samples? As you know, this is extremely common outside of large scale projects like ENCODE and Blueprint: A biologist starts with three cell cultures, runs RNA-seq, knocks out a gene in them, does RNA-seq, treats with some drug and does Chip-Seq on the gene-knockout-cell culture. In your data model, these samples would all be independent, and the relationships between them, the tree structure, are lost. To me it seems that your data model doesn't support inheritance or relationships between samples or between experiments, is this correct?\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] }, { "id": "87124", "date": "24 Jun 2021", "name": "Granger Sutton", "expertise": [ "Reviewer Expertise pan-genome analysis", "whole genome shotgun assembly", "genome annotation", "homology search", "multiple sequence alignment" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well written opinion article describing a proposed set of standards for genomic track metadata. As the authors point out in the conclusion this is merely a first step in the process towards community acceptance but without draft proposals such as this there is nothing to work towards. I am not an expert on many aspects of the proposed standards but am certainly familiar with many of the issues and use cases. This seems like a reasonable effort at starting the discussion. The proposal is enhanced by the prototype use of the standard and the availability of tools for using it.\nI had two minor quibbles with the article. First, it is not clear to me that “A survey of metadata keys available on the track hubs suggests a great disparity in usage as provided in Table 1.” is correct. Earlier in the article Table 1 was described to contain “Domain-specific metadata standards exist that guide current efforts to annotate genomic datasets (see Table 1 for a summary of object types across these data models and their required attributes).” which does appear to be correct. Perhaps both are correct, but this needs to be explained if so. I think a better description of what is in Table 1 needs to occur earlier in the article.\nSecond, “This testing phase clarified a need for an automated intermediate step between metadata curation and consumption, which in most cases simply meant to augment resolvable identifiers with human readable versions of the same.” needs to be clarified perhaps with an example.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-268
https://f1000research.com/articles/10-265/v1	01 Apr 21	{ "type": "Brief Report", "title": "Syntax-semantics interactions – seeking evidence from a synchronic analysis of 38 languages", "authors": [ "Tom S Juzek", "Yuri Bizzoni" ], "abstract": "The notion that, to facilitate processing, as semantic complexity increases, syntactic complexity decreases, follows from various linguistic theories. This brief report presents the results of testing that notion, by analysing synchronic data from 38languages and correlating canonical measures of semantic and syntactic difficulty. We expected an overall positive tendency. However, the results came out mixed to negative. There is a notable degree of variation and there are no clear tendencies within language families. After detailing the theoretic and cognitive reasons that support the original hypothesis, we conclude with a short discussion about the potential causes and implications of our findings. A possible interpretation is that the interaction we are looking for is more subtle than one might have assumed.", "keywords": [ "cognitive linguistics", "corpus analysis", "syntax", "semantics", "syntax-semantics interactions", "efficient communication", "dependency grammars", "Universal Dependencies", "null results" ], "content": "Introduction\n\nThe syntactic complexity and semantic difficulty of a sentence are two different aspects of language, each requiring an independent amount of processing effort.1 Many linguists also assume that the interplay between different linguistic levels can facilitate efficient communication.2\n\nA relatively popular assumption is that syntax facilitates semantics.1,3–5 Several linguists and philosophers of language assume that grammar was optimized for rational and efficient communication.6 Various recent studies that have looked into the question from an evolutionary and typological viewpoint also support the idea that the drive for communicative efficiency has shaped syntactic features such as subject-verb-object order7 or average dependency length,8 and semantic domains such as numeral systems9 and colour terms.10 Concerning grammar, it seems safe to suppose that denser semantic content tends to reduce syntactic complexity. This notion is an explicit part of some linguistic theories, e.g., systemic functional grammar,11 and is in harmony with other theories, e.g., the uniform information density hypothesis.12–14 However, despite an increasing body of research, a large-scale study of synchronic data, comparing a semantic with a syntactic analysis, is still missing.\n\nUnder the view that syntax facilitates semantics, it is plausible to assume that on average writers and speakers will deliver difficult semantics via simpler syntax and simplify semantics in the presence of difficult syntax in order to ensure a healthy level of communicative complexity - price the risk of misunderstanding. That is, we expect a tendentially inverse relationship between semantic and syntactic complexity. Whether real data corroborates this expectation is the research question framing this report. We elaborate it through two competing hypotheses.\n\nH0: There is no systematic interaction between semantic complexity and syntactic complexity.\n\nH1: There is an inverse relationship between semantic complexity and syntactic complexity: as lexical semantics complexity increases, syntactic complexity decreases, and vice versa.\n\nA repository containing all files relevant for replication of this research can be found online.33,34\n\n\nMethods\n\nTo test the hypotheses, we used the corpora made available through the Universal Dependencies (UD) projecta, accessed in June 2020. The UD corpora are, especially for large languages, a mix of different registers and domains, ranging from blog posts to legal texts, the vast majority of which comes from contemporary sources. Critically, the corpora contain manually annotated syntactic information in the form of dependency relations. The fact that syntactic annotation was done manually and not automatically is beneficial for our goals, since it removes the risk of systematic mistakes introduced by automatic syntactic annotation. For any language with more than one (sub)corpus, we merged the corpora and only included languages for which we have more than 0.1m tokens in total. Further, we only considered languages that have their lemmas specified, which leaves us with the languages we list in Table 1.\n\nAn asterisk indicates a p<=0.05. The median sentence length for any given language is in the “med. len.” column. “N” is the number of sentences analyzed. “r long” is the correlation coefficient for twice the median sentence length.\n\nWe measured semantic complexity with a canonical and straightforward measure: the sum of the log frequencies of a sentence’s lemmasb. The inverse correlation between word frequency and its processing difficulty is widely known,15,16 while lemmas and inflected forms do not appear to behave very differently in terms of distribution.17 If readers encounter a particular word several times, they become more effective in processing it both at the orthographic and semantic levels; and the more they encounter the word, the more effective they become. Thus, all other things being equal, a sentence’s sum of log lemma frequency should be a proxy for its processing cost. The reason why we did not opt for sequence surprisal or perplexity, for example, is that sequences longer than one word necessarily reflect syntactic behaviours. That is, at an n-gram level, surprisal and perplexity contain both semantic and syntactic information. They are functionally equivalent to the frequency measure at a unigram level, so we use the latter as our semantic measure.\n\nWe used the UD framework to measure syntactic complexity. The UD framework expresses syntactic relations through dependencies: each element depends on another element, its head.18 In UD, the head is the semantically most salient element, and the dependent modifies the head. The top-level head is the root of a sequence, typically the main verb of the matrix clause. Critical to our syntactic measure is the notion of dependency length. For each element, we can calculate the distance to its head, where each intervening element increases the distance by one, and for each sentence, we can add up the distances for a sentence’s elements, which results in the sum of dependency lengths. Figure 1 illustrates a dependency analysis; the sum of distances in that example is 6 (2+1+2+1). The measure is taken from the literature.2,19–21 We consider this an intuitive measure: all other things being equal, sentences with shorter dependency lengths will be easier to process.2,22\n\n(Created with spaCy: https://spacy.io/.)\n\nSince the relationship between sentence length and summed dependency distances is not necessarily linear,2 there is no simple way to normalise for sentence length. Thus, to remove the risk of spurious sentence length effects, we computed our correlation only at the median sentence length per language, excluding punctuation.c For example, the median sentence length for English is 12 tokens, while for Dutch, it is 6. Another aspect we controlled for is word length. Word length is known to roughly correlate with information content,23 so for any median token length, we further extracted the median character length and considered a range of +/-10%. For instance, the median character length for English sentences of 12 tokens is 47 characters, so we only considered sentences with 12 tokens that are within a range of 42 to 52 characters. Since the median sentence length is relatively short for some languages, we also analysed longer sentences, viz. twice the median sentence length, which is the ‘long’ condition.\n\nWe calculated the Pearson correlation coefficient between the summed logged frequencies and the summed dependency distances for each language, using R.24 For our two measures, positive correlation coefficients are evidence for H1: We expect low frequencies (high semantic complexity) to correlate with shortened dependency lengths (low syntactic complexity).\n\n\nResults and discussion\n\nThe results are given in Table 1 and are in parts illustrated in Figure 2. Due to space limitations, we omitted the N for the long condition. In total, we analyzed about 31.6k sentences in the normal condition and about 16.1k sentences in the long condition.\n\nEach dot represents the results for a certain sentence. Within a language, all sentences are of the same token length. (Created with R.24)\n\nOur results are mixed-to-negative and seem to be language-dependent. There is a rather high variance centred around a mean near zero: 21 languages display the expected positive correlations, 9 of which are significant, but 17 languages display negative correlations, 5 of which are significant. We cannot interpret these findings as evidence for H1, and while it is difficult to affirm H0, we cannot reject it.\n\nTaken at face value, this would mean that in some languages, an increase in semantic complexity implies an increase in syntactic complexity, which seems implausible, also since there is quite some variation within language groups, e.g., Slovenian and Bulgarian or Swedish and Dutch. There could be various other causes for the observed variation and the lack of a systematic correlation as discussed below.\n\nWe used a canonical, easy to compute, and corpus-dependent semantic measure, amply verified in linguistics, information theory, and cognitive science. While it is a proxy for semantic complexity, as such it leaves room for improvement, we find it unlikely that the mixed results we observe are entirely due to our measure of complexity.\n\nOur syntactic measure depends on the UD framework’s validity, which aims to be language universal.18 We cannot exclude that there are inconsistencies in theoretical choices across languages, e.g., how prepositions or relativizers are attached.25 An in-depth qualitative analysis is needed to follow up on this, and those choices might have contributed to some variance. The extensive amount of work gone into the formulation of the UD framework, together with the efforts made to ensure the consistency of UD syntactic annotations across different languages, makes us think that internal inconsistencies should not shift the mean to a degree such that it conceals a real effect.\n\nSpecific registers, or even domains, might be characterised by an unusual complexity of both the semantic and the syntactic side, or have a technical vocabulary that becomes “simple” to their usual readers, leaving room for more syntactic complexity. However, the fact that we draw our semantic measures from the corpora themselves should mitigate such an effect: if a rare semantic unit frequently appears in one of our corpora, its frequency increases, and its complexity level decreases. While differences in the corpora’s makeup could cause parts of the observed variance, there is no apparent reason why they should have caused the mean to shift towards zero.\n\nOther factors that we could not account for could be at play. This concerns pragmatic aspects26 or even morpho-phonetic aspects of the lexicon.27–30 Another source of noise in our data could be that syntactic structures can increase in complexity to a good extent without requiring any semantic simplification or even favoring semantic complexity.31\n\nWe have identified some factors that could cause the observed variance. However, if the interaction between syntax and semantics was as strong as we assumed, then there should have been a measurable shift towards a positive mean in most languages. That means that there should have been some signal from the data at hand “shining through” the noise. Nonetheless, the results are not evidence of our H0: the absence of evidence is not the evidence of absence.32 A practical takeaway could be that the interaction between syntax and semantics is more complex and more subtle than we sometimes conceived it to be, certainly at a synchronic level.\n\n\nConclusions\n\nThis brief report explored in how far syntax and semantics interact in synchronic, large-scale data from 38 languages. To compute syntactic and semantic complexity, we relied on two common measures: dependency length and unigram frequency. These measures are only one way to approach the problem, but their simplicity and empirical robustness make them powerful and unassuming proxies, widely employed in computational and cognitive linguistics. Against our expectation, the analysis did not produce a widespread positive correlation between the two measures that would express an inverse correlation between syntactic and semantic complexity. In many of the languages represented in our data, even when the semantic processing load is high, its syntactic complexity does not appear systematically lowered. Since we used the most straightforward method we conceived of to test our hypothesis, we think that if the effect we hypothesised was obvious and consistent across languages, it should have been visible with the methodology we used, i.e., the mean of all correlations should have had a clear positive tendency. Since this is not the case, our results hint at the possibility that the interaction we are looking for is more subtle than one could have assumed. We find our negative results surprising, and we hope that our report stimulates both discussion and further research.\n\n\nData availability\n\nZenodo: Syntax-semantics interactions – seeking evidence froma synchronic analysis of 38 languages: dataset repository. https://doi.org/10.5281/zenodo.4542536.\n\nThis project contains the following underlying data:\n\n- A subset of the Universal Dependencies Corpora v2.6 used in this study.\n\nApache 2.0 license, with the Universal Dependency Consortium holding the copyright.\n\nZenodo: Syntax-semantics interactions – seeking evidence froma synchronic analysis of 38 languages: scripts repository. https://zenodo.org/record/4643152.\n\nThis project contains the following extended data:\n\n- The scripts used for data preparation and analysis.\n\nThe scripts are available under a CC0 license (”No rights reserved”). While the scripts modify the data sets, the modified data sets continue to be subject to the same Apache 2.0 license as the original data sets.\n\n\nAuthor contributions\n\nYB and TSJ co-designed the study. TSJ coded the analysis.\n\n\nCompeting interests\n\nThere are no competing interests.", "appendix": "Footnotes\n\na https://universaldependencies.org/\n\nb We log the absolute frequencies. Since we apply the measure only to the corpus from which we take it, this does not lead to distortions.\n\nc As a test, we computed this correlation for all sentence lengths in English, finding consistent results.\n\n\nReferences\n\nOstrin RK, Tyler LK: Dissociations of lexical function: Semantics, syntax, and morphology. Cogn Neuropsychol. 1995; 12(4): 345–389. Publisher Full Text\n\nGibson E, Futrell R, Piantadosi S, et al.: How Efficiency Shapes Human Language. Trends Cogn Sci. 2019; 23(5): 389–407. PubMed Abstract \| Publisher Full Text\n\nBoland JE: The relationship between syntactic and semantic processes in sentence comprehension. Lang Cogn Processes. 1997; 12(4): 423–484. Publisher Full Text\n\nAinsworth-Darnell K, Shulman HG, Boland JE: Dissociating brain responses to syntactic and semantic anomalies: Evidence from event-related potentials. J Mem Lang. 1998; 38(1): 112–130. Publisher Full Text\n\nJohns CL, Campanelli L, Kush D, et al.: Individual differences in combinatorial semantic processing: Skilled comprehension facilitates complement coercion during sentence comprehension.2019. Publisher Full Text\n\nFrank AF, Jaeger TF: Speaking rationally: Uniform information density as an optimal strategy for language production. In Proceedings of the annual meeting of the cognitive science society 2008; volume 30.\n\nHahn M, Jurafsky D, Futrell R: Universals of word order reflect optimization of grammars for efficient communication. Proc Natl Acad Sci U S A. 2020; 117(5): 2347–2353. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFutrell R, Levy RP, Gibson E: Dependency locality as an explanatory principle for word order. Language. 2020; 96(2): 371–412. Publisher Full Text\n\nXu Y, Liu E, Regier T: Numeral systems across languages support efficient communication: From approximate numerosity to recursion. Open Mind. 2014: pages 1–14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSteinert-Threlkeld S, Szymanik J: Ease of learning explains semantic universals. Cognition. 2020; 195: 104076. PubMed Abstract \| Publisher Full Text\n\nHalliday MAK: Introduction: On the “architecture” of human language. In: Webster J, editor, On lang ling. London, New York: Continuum; 2003; chapter 1. : pages 1–30.\n\nAylett M, Turk A: The smooth signal redundancy hypothesis: A functional explanation for relationships between redundancy, prosodic prominence, and duration in spontaneous speech. Lang Speech. 2004; 47(1): 31–56. PubMed Abstract \| Publisher Full Text\n\nJaeger TF, Levy RP: Speakers optimize information density through syntactic reduction. In: Schölkopf B, Platt JC, Hoffman T, editors, Advances in Neural Information Processing Systems 19. 2007: pages 849–856. MIT Press.\n\nJaeger TF: Redundancy and reduction: Speakers manage syntactic information density. Cogn psychol. 2010; 61(1): 23–62. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKuperman V, Van Dyke JA: Reassessing word frequency as a determinant of word recognition for skilled and unskilled readers. J Exp Psychol Hum Percept Perform. 2013; 39(3): 802. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPiantadosi ST: Zipf’s word frequency law in natural language: A critical review and future directions. Psychon Bull Rev. 2014; 21(5): 1112–1130. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCorral A, Boleda G, Cancho RF: Zipf’s law for word frequencies: Word forms versus lemmas in long texts. PloS One. 2015; 10(7): e0129031. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNivre J, Abrams M, Agić Z, et al.: Universal Dependencies 2.4.2019. Reference Source\n\nFutrell R, Mahowald K, Gibson E: Large-Scale Evidence of Dependency Length Minimization in 37 Languages. Proc Natl Acad Sci U S A. 2015; volume 112: pages 10336–10341. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGibson E: The dependency locality theory: A distance-based theory of linguistic complexity. Image, language, brain. 2000: 95–126.\n\nLiu H: Dependency Distance as a Metric of Language Comprehension Difficulty. J Cogn Sci. 2008; 9: 159–191. Publisher Full Text\n\nHawkins JA: Efficiency and Complexity in Grammars. Oxford: Oxford University Press; 2004.\n\nLewis ML, Frank MC: The length of words reflects their conceptual complexity. Cognition. 2016; 153: 182–195. PubMed Abstract \| Publisher Full Text\n\nR Core Team: R: A Language and Environment for Statistical Computing. Vienna, Austria: R Foundation for Statistical Computing; 2020. Reference Source\n\nOsborne T, Gerdes K: The status of function words in dependency grammar: A critique of universal dependencies (ud). Glossa. 2019. Publisher Full Text\n\nKuperberg GR, McGuire PK, Bullmore ET, et al.: Common and distinct neural substrates for pragmatic, semantic, and syntactic processing of spoken sentences: an fmri study. J Cogn Neurosci. 2000; 12(2): 321–341. PubMed Abstract \| Publisher Full Text\n\nGraves WW, Grabowski TJ, Mehta S, et al.: A neural signature of phonological access: distinguishing the effects of word frequency from familiarity and length in overt picture naming. J Cogn Neurosci. 2007; 19(4): 617–631. PubMed Abstract \| Publisher Full Text\n\nRothermich K, Schmidt-Kassow M, Kotz SA: Rhythm’s gonna get you: Regular meter facilitates semantic sentence processing. Neuropsychologia. 2012; 50(2): 232–244. PubMed Abstract \| Publisher Full Text\n\nCanette L-H, Lalitte P, Bedoin N, et al.: Rhythmic and textural musical sequences differently influence syntax and semantic processing in children. J Exp Child Psychol. 2020; 191: 104711. PubMed Abstract \| Publisher Full Text\n\nKing A, Wedel A: Greater early disambiguating information for less-probable words: The lexicon is shaped by incremental processing. Open Mind. 2020; 4: 1–12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchoenemann PT: Syntax as an emergent characteristic of the evolution of semantic complexity. Minds Machines. 1999; 9(3): 309–346. Publisher Full Text\n\nAltman DG, Bland JM: Statistics notes: Absence of evidence is not evidence of absence. Bmj. 1995; 311(7003): 485. Publisher Full Text\n\nJuzek TS, Bizzoni Y: Syntax-semantics interactions – seeking evidence froma synchronic analysis of 38 languages: dataset repository (Version 1.0) [Data set]. Zenodo. 2021. Publisher Full Text\n\nJuzek TS, Bizzoni Y: Syntax-semantics interactions – seeking evidence froma synchronic analysis of 38 languages: scripts repository (Version v1.1). Zenodo. 2021, February 16. Publisher Full Text" }	[ { "id": "82680", "date": "10 Jun 2021", "name": "Chunshan Xu", "expertise": [ "Reviewer Expertise Cognitive linguistics and dependency grammar" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting and original work concerning the relation between syntax and semantics. But, there are some issues worthy of further discussion or clarification.\n\nFirst of all, syntax facilitates semantics. This is true. But this does not mean that \"writers and speakers will deliver difficult semantics via simpler syntax and simplify semantics in the presence of difficult syntax\". In fact, the complex syntax is probably a result of pressure for communication of complex semantic message.\n\nSecondly, it is very difficulty to quantitatively define semantic difficulty. The authors have used the sum of the log frequencies of a sentence’s lemmas. This is more a measure of expectation, or, information. In fact, in conveying complex semantic message, it is important to keep the information content at a proper level at any time: it can neither be too high nor too low (uniform information density hypothesis). The relatively even distribution of information might be one of tasks of syntax. Zipf's law of word frequency distribution is probably one piece of evidence.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "96455", "date": "23 Nov 2021", "name": "Diego Frassinelli", "expertise": [ "Reviewer Expertise Computational linguistics", "corpus analysis", "cognitive science" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this paper, the authors test a very straightforward (at least in theory) and interesting assumption that there should be an inverse correlation between the complexity of syntax vs. semantics in language. They perform a systematic analysis of text in 38 different languages. They quantify semantic complexity as uni-gram frequency and syntactic complexity as dependency length (distance from the head). The results are neither confirming nor completely negating the predictions based on the literature and are very language-dependent.\n\nWhat I liked:\n\nAll in all, this is a simple interesting research question that can (and should) be addressed from a corpus analysis perspective. It is nice to see the attempt of analyzing very different languages and trying to identify relationships between them. Moreover, the code used in this study is available and allows, as far as I can say, full replicability.\n\nWhat could be improved:\nI think this article needs a bit of extra work in order to be indexed. Unfortunately, at the moment, it is not really clear if we can rely on the results reported or if what we see is simply due to other strong cofounding elements. Below are some comments:\nI find the statement that complex syntax calls for simple semantics and vice-versa a bit of an overgeneralization. In many domains (in particular web data), I would expect a simple-simple interaction instead.\n\nThe authors correctly indicate that frequency is a good indicator of processing cost. However, processing cost does not exactly correspond to semantic complexity: such complexity is way more multifaceted and, I feel, not really captured by frequency alone.\n\nMoreover, focusing on the semantics of single words (treated as out of context entities) is probably an oversimplification: I understand that the inclusion of contextualized measures would indirectly capture syntactic information too, but not including this information makes this measure probably too simple to capture semantic complexity.\n\nOne possible confounding variable that, in my opinion, needs to be directly addressed in the experiment is the effect of genre/domain of the text used. At the moment the effect is only briefly described as a possible cause, but it should really be included in the statistical analysis. Very specific domains (e.g., legal text) follow specific styles that differ from all the rest and this can add a lot of \"noise\" to the data. This becomes even more of an issue once the study tries to compare different languages based on corpora that, as far as I understand, are not balanced in any systematic way.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-265
https://f1000research.com/articles/10-264/v1	01 Apr 21	{ "type": "Method Article", "title": "COVID-19 and digital transformation: developing an open experimental testbed for sustainable and innovative environments using Fuzzy Cognitive Maps", "authors": [ "Wolfgang Höhl" ], "abstract": "This paper sketches a new approach using Fuzzy Cognitive Maps (FCMs) to operably map and simulate digital transformation in architecture and urban planning. Today these processes are poorly understood. Many current studies on digital transformation are only treating questions of economic efficiency. Sustainability and social impact only play a minor role. Decisive definitions, concepts and terms stay unclear. Therefore this paper develops an open experimental testbed for sustainable and innovative environments (ETSIE) for three different digital transformation scenarios using FCMs. A traditional growth-oriented scenario, a COVID-19 scenario and an innovative and sustainable COVID-19 scenario are modeled and tested. All three scenarios have the same number of components, connections and the same driver components. Only the initial state vectors are different and the internal correlations are weighted differently. This allows for comparing all three scenarios on an equal basis. The Mental Modeler software is used. This paper presents one of the first applications of FCMs in the context of digital transformation. It is shown that the traditional growth-oriented scenario is structurally very similar to the current COVID-19 scenario. The current pandemic is able to accelerate digital transformation to a certain extent. But the pandemic does not guarantee for a distinct sustainable and innovative future development. Only by changing the initial state vectors and the weights of the connections an innovative and sustainable turnaround in a third scenario becomes possible.", "keywords": [ "soft computing", "fuzzy cognitive maps", "digital transformation", "COVID-19", "decision making", "sustainability", "integrated world system modeling" ], "content": "Introduction\n\nYou probably all know this interactive graphic very well. Figure 1 shows a real-time visualization of the worldwide spread of the corona virus by the Johns Hopkins University in Baltimore.1 This interactive map was created with ArcGIS Online. As an architect and planner you probably know similar GIS software that is used in comparable cases. This graphic is a good example for emerging technologies in the current digital transformation. The current pandemic is accelerating digital transformation. This is not only the opinion of German Federal Minister Andreas Scheuer.2 The President of the German Conference of Ministers of Education and Cultural Affairs, Dr. Stefanie Hubig, also expects a boost for digital transformation in schools.3 But there are also critical voices. In a petition to the German ministers of education, the Munich teacher Tina Uthoff calls for “an end to distance learning”.4 Not all families are able to provide the same level of care and are under great pressure. But there are also other interesting solutions. A Munich wholesale market for gastronomy and retail now supplies food to senior citizens and social food banks with the help of taxi drivers. Logistics software provides the optimized routes for the taxi drivers. The remarkable and decisive factor here is not only using innovative technologies but an innovative idea. Much more it is a specific way to think about solutions, a special “thought style”, a guiding mission statement and a clear direction. An effective push always needs a clear direction. The whole process of digital transformation today is very poorly understood. Therefore this paper sketches a new approach using Fuzzy Cognitive Maps (FCMs) to operably map and simulate the process of digital transformation in architecture and urban planning. Fuzzy Cognitive Maps (FCMs) belong to the so-called soft computing techniques, such as fuzzy logic, neural network theory, genetic algorithms and probabilistic reasoning. Zadeh5,6 defines soft computing as an efficient technique that incorporates human knowledge effectively, deals with imprecision and uncertainty and learns to adapt to unknown or changing environments for better performance. Bonissone7 and Jain8 mention that soft computing has been successfully applied in many scientific areas such as in engineering, medicine, information systems, business, political and social sciences. FCMs are flexible tools that have been applied in different contexts9 including environmental assessment,10-12 engineering and technological management13 and energy.14 Papageorgiou and Groumpos15 especially consider FCMs to be capable to deal with situations where the human reasoning process includes uncertain descriptions. FCMs are usually used for modelling specialist knowledge. For data gathering and model building expert interviews and expert opinions are used. Also the delphi method or similar methods can be applied in this initial stage. Secondly FCMs include important modeling means for describing particular domains showing the concepts (variables) and the relationships (connections) between them. For modeling and simulation usually specialized FCM software is used. FCMs are described as a well-established artificial intelligence technique that incorporates ideas from artificial neural networks and fuzzy logic. Thus they are an attractive modeling approach that encompasses advantageous features. On FCMs the following weaknesses can be detected: the main deficiencies of FCMs are the critical dependence on the initial expert’s beliefs, the recalculation of the weights corresponding to each concept every time a new strategy is adopted and the potential convergence to undesired equilibrium states. In order to update the initial knowledge of human experts and to combine the human experts’ structural knowledge with training from data, specific automated learning methodologies for FCMs my be applied. This paper presents one of the first applications of FCMs in the context of digital transformation. In the current discussion, a fundamental clarification of the terms and their correlations in digital transformation seems to make sense. Thus this paper provides an overview of literature. In this fundamental discourse, fourteen up-to-date studies on digital transformation in architecture and urban planning17-30 and twelve studies from the fields of computer science, philosophy of science and media ethics have been searched for stakeholders, specific terms or concepts and correlations in digital transformation.31-42 Terms are clarified, technologies and trends can be identified. The specific “thought styles” and paradigms of the stakeholders are discovered. These elements are then operably mapped and represented in a suitable, integrated model using FCMs. A value added open experimental testbed for sustainable and innovative environments (ETSIE) is developed. Three different digital transformation scenarios are designed. A traditional growth-oriented scenario, a COVID-19 scenario and an innovative and sustainable COVID-19 scenario are modeled and tested. All three scenarios have the same number of components, connections and the same driver components. Only the initial state vectors are different and the internal correlations are weighted differently. This allows for comparing all three scenarios on an equal basis. The Mental Modeler software is used.16 This paper develops the value added ETSIE model as a general framework using FCMs, provides a discussion integrating an overview of literature and highlights directions for future inquiry, but it is not expected to collect extensive empirical data. It is shown, that the traditional growth-oriented scenario is structurally very similar to the current COVID-19 scenario. The current pandemic is able to accelerate digital transformation to a certain extent. But the pandemic does not guarantee for a distinct sustainable and innovative future development. Only by changing the initial state vectors and the weights of the connections an innovative and sustainable turnaround in a third scenario becomes possible.\n\n\nLiterature review\n\nThis chapter presents a fundamental discourse, fourteen up-to-date studies on digital transformation in architecture and urban planning17-30 and twelve studies from the fields of computer science, philosophy of science and media ethics31-42 have been searched for stakeholders, causes and key drivers, technologies and trends in digital transformation. Terms or concepts are clarified, technologies, trends and five indicators of sustainable development could be identified. Specific “thought styles” and paradigms of the stakeholders are discovered.\n\nAccording to Horx,17 Widmann,18 Detting19 and von der Gracht20 there are two general characteristics of the corona crisis: on the one hand the novelty of the corona virus and on the other hand the relatively slow course of the crisis compared to other natural disasters. Horx sees the corona crisis as “ ... a general slowdown of our world culture”, which affects both globalization and our forms of communication. In his opinion, this slowdown will continue after the crisis. “If you take stock, this crisis is a general slowdown of our world culture. This applies to globalization and to our forms of communication. It is a slowdown that will remain.”17 On the contrary for Widmann the corona crisis is not a natural disaster in the conventional sense.18 According to all we know today, this pandemic is of natural origin. However, pandemics distinguish between people and material assets, and they always affect the socially weak most severely. However, the boundaries to natural disaster remain blurred. Detting outlines a possible more resilient and robust future for society between the polar risks of total interstate and social isolation and the chance of a new sustainable and glocal social-ecological market economy.19 Depending on the duration of the crisis, von der Gracht sketches four polar scenarios between very moderate and very drastic economic and social consequences.20 The strong isolation, social conflicts and the virtualization of many areas of life face a rapid recovery and a return to what he calls “normal operations”. A positive outlook for a new and innovative social scenario is unfortunately missing here. COVID-19 is novel and cannot be fought with conventional means. Innovative ideas and technologies must be developed. Due to economic interests, a certain time pressure arises, which pushes the research and development of new technologies. So COVID-19 is an accelerator of digitalization and technological development, in the same sense as other natural disasters can be. Thus the corona crisis can be described as a natural disaster in slow motion. Its relatively slow or time-stretched course compared to other natural disasters allows for a coordinated, reasonable and gradual technological response. The associated deceleration and slowing down of everyday life during the first corona lockdown is certainly still well remembered by us all. The reduction of many growth-driven processes during the Corona crisis thus also led to a certain social and ecological sustainability.\n\nAll industries are directly affected by digital transformation. The processes only differ in speed and intensity. Thierstein describes digital transformation as global and all-encompassing: “Digital transformation permeates all ways of life ....”21 Goger et al. describe digital transformation as a cross-cutting issue that cuts across all areas of society.22 The industries only have a different digitization speed. Roland Berger believe that the construction industry is less affected by the corona crisis, as are mechanical engineering and pharmaceutical and medical technology.23 They see most other industries as being heavily affected, such as airlines, tourism and travel, trade, financing, oil and gas, automotive and logistics. After examining the aforementioned available literature8-33 four different stakeholders groups can be identified: (P) politics, (R) research and development, (E) economy and (S) civil society as shown in Table 1.\n\nA study by IE.F and Roland Berger defines the following drivers for a successful digital transformation as shown in Table 2.24 These drivers can be easily assigned to the four different stakeholders groups.\n\nGoger et al. mention the following components as key drivers of digital transformation in architecture and urban planning as shown in Table 3:22\n\nAccording to a study by the “Münchner Kreis”, product and process quality (79%) and qualified employees training (78%) are currently considered the most important success factors in future industrial manufacturing.24 Alain Thierstein also mentions the importance of the structure of creative and productive processes. He emphasizes the technological competence of the individual (digital literacy), which is necessary to take responsibility, to evaluate situations and to acquire the new digital environment.21 Table 4 shows the collected causes and drivers from the these two references:\n\nRoland Berger sees digitization in the workplace and the home office as a direct effect of increasing digital transformation.23 Thus Berger confirms the interconnections between the use of digital technology, user behavior and mobility (e.g. home office, digital collaboration). Goger et al. estimate that there is a 59% probability of substituting construction occupations with Industry 4.0. At least they call it “degree of automation” or “probability of automation” and present their “vision of digital construction”.22 During the early phases of development, better variant studies for decision making become possible through visualization and simulation, better and significantly more transparent information exchange. Central digital twins of the building and applications of BIM in integral planning are getting in the field of vision, as well as processes for automated quality assurance and compliance with building standards, simplified tendering on a digital basis and fully automated reading of masses and quantities. During construction, improved logistics through RFID tracking with location allocation would be possible. Digital recording of delivery bills and material parameters and a complete documentation would digitally supplement the construction progress. Simplified surveys could be provided through drone flights. Innovative manufacturing processes could support or even replace conventional procedures. IoT and Big Data will be deployed during building use for automatic building data collection, automatic ventilation and building climate control. During demolition, the building can serve as a raw material store (urban mining). Through a digitally transparent process, all quantities and materials can be known in advance. Katz et al. describe digitization speed as a composite factor of affordability, infrastructure investment, network access, capacity, usage and human capital.25 As digitization speed can not be assigned to one stakeholder only, a new category of indicators for system behaviour is created. With the degree of automation and the digitization speed the authors name two important indicators for the social sustainability of digital transformation. Table 5 shows the extended list of stakeholders and indicators.\n\nThe “vision of digital construction” has a high social relevance in addition to its economic relevance. Schüller developed the term “digital literacy”.27 He also names it among the so-called success factors of the digital transformation. The speed of digitization in the respective industries will depend on the digital literacy of those involved in the process and the quality of products and services. Simondon warns against such purely economically driven automation.28 He describes automation by the well-known image of a fully automatic and autonomous robot. Simondon sees in it only an abstract mythical object, without any relevance for a practical and really innovative technological development. “Automatism and its use in the form of the industrial organization called automation has far more of an economic or social than a technical meaning.”28 In order to ensure a functioning human-machine interaction, Simondon advocates “open machines” with a certain “margin of uncertainty”. Developments such as artificial intelligence, algorithms for the individualization and personalization of interfaces or even autonomous driving could easily be assigned to this fully digitalized automation. According to Baumanns, the interdisciplinary collaboration across all phases of a building’s life cycle (design, construction, operation) also still offers great potential for digital transformation. This includes a central, digital building model and digital, interdisciplinary communication and coordination. Baumanns defines the following megatrends29 as shown in Table 6. As growth drivers the authors describe residential construction, infrastructure and transport. The general construction volume is currently benefiting from low loan and interest rates. As competitive advantages for companies he sees the areas of Smart Home, Smart Building and Building Information Modeling (BIM). As current opportunities he mentions globalization, specialization and expansion of the company’s own portfolio in the existing value chain and the increasing use of digital technology. The greatest potential he assumes in the field of logistics, in the digital collection and analysis of data and in the automation of construction work. He names five phases: logistics, procurement, production, marketing/sales and after sales.\n\nAnalogous to Industry 4.0, Goger et al. coined the term “Construction 4.0”.22 The Digital Roadmap Austria identifies ten technology areas with enormous development potential that could also be of significance for architecture and urban planning.30 A study by BRZ Deutschland GmbH. names the following six IT-trends in the construction industry.31 However, in these studies there is no distinction between conventional and sustainable technologies. Some of the technologies mentioned, such as block chain technology, are currently heavily criticized, because of their disproportionate energy consumption. The sustainability of IoT is also under current discussion. The integrated IT and the lack of software updates for intelligent household appliances could lead to a shorter lifetime of usual household appliances such as refrigerators. Moreover today there is no clear distinction and recommendation between sustainable technologies and non-sustainable technologies. Additionally there are currently no comparable indicators of the systemic energy efficiency of the individual technologies. This could be ensured by a technology-specific ecological footprint. To take these factors into account, two further indicators will be introduced to assess the sustainability of the overall system: primary energy consumption and the ratio of sustainable processes and technologies to the total amount of digital processes and technologies as shown in Table 7. To my opinion, the “virtual project space” and “cloud computing” can be described more as distinct technologies and less as a trend. Established in science, the term “Collaborative Virtual Environments” (CVE) has long been used to describe these technical solutions. The same applies to BIM. I would only use the term trend in connection with an overarching scenario based on a specific thought style, mindset or paradigm.\n\nDigitization, digital transformation or innovation?\n\nMany authors mention digitization itself as a driver for major changes in work processes. It would be much more accurate to name technological change through research and development as a driver of digitization. Thus research and development appeared as a stakeholder on the scene. The newly developed technology and the associated research and development (not digitization or digital transformation) are the drivers. To a large extent new technological developments lead to changes in work processes. Digital transformation can be better described as the systemic end result or as a significant scenario in this macrosocietal transformation. Digitization and technology are also listed as trends in many studies. What is missing here is a precise distinction between digitization (or digital transformation) as a cross-system scenario on the one hand and digital technology as a subordinate systemic element resulting from research and development on the other. Just as BIM, smart homes and smart buildings can be better described as technologies rather than as trends. Digitization and digital transformation are not used as congruent terms in this paper. Digital transformation better desribes the whole process. While digitization can usually be understood as the “conversion of analogue quantities into digital ones”. But neither digitization nor digital transformation does not necessarily mean innovation. The digitization of an existing process does not necessarily have to be innovative per se. Purely economic and efficiency-oriented digitization is the adaptation of an existing process to modern technology. However, this does not necessarily make the process new and certainly not sustainable. Innovation always means a previously unknown, new solution to a problem. The innovative idea is also fundamental to innovation, which then leads to a new solution by recombining existing resources. At the beginning of an innovation there is always an idea and a new mindset, a new way of thinking about things. The implementation into a suitable technological solution is subordinated.\n\nSustainability\n\nIn the examined literature, sustainability is often used as an ambiguous term or concept. For example Baumanns cites sustainability itself as a current megatrend.29 Thus sustainability appears in a completely different context than in IE.F and Roland Berger24 and Goger et al.22 There sustainability is mentioned as a trend. In many studies sustainability is seen as a way of thinking, a thought style or a paradigm. It can also be interpreted as a social factor, such as a sustainable lifestyle. Or it can be interpreted as an economic factor such as a new sustainable business and corporate culture. Sustainability is often mentioned in connection with an economically efficiency-oriented process optimization. Many experts also expect sustainability to have positive effects on future energy and resource consumption. Last but not least it could stand for either a sustainable research paradigm or a sustainable political “thought style”. The term sustainability can also be interpreted in a political sense as the amount of political actions aiming climate protection. Thus sustainability itself cannot precisely be assigned to one stakeholders group only. The term finds correspondences in all four stakeholders groups as a “thought style” or a specific way of thinking. So it will reappear again as a political “thought style”, a “research paradigm”, a public “thought style” and an “economic business and corporate culture”. It also can be defined as another separate concept, such as political or economical actions for climate protection. Sustainability also can be seen as a quality of the whole system behaviour, when we think of technologies and trends. So indicators such as primary energy consumption, the degree of automation, digitization speed and the ratio of sustainable processes and technologies to the total amount of digital processes and technologies can be seen strongly related to the main concept of sustainability as well.\n\nIn the aforementioned sections, another group of causes and drivers of the digital transformation emerges. It is the group of thought styles, paradigms, ways of thinking or reasoning. This group will be explained in more detail in the next section. Ian Hacking coined the term “style of reasoning” in connection with the continuous change in mission statements.32 He essentially followed Alistair Crombie (1915-1996), who claimed that there are different scientific methods of knowledge that have emerged in certain areas of human history. Ludwik Fleck developed the analogous concept of “thinking styles”.33 Rudolf S. Kuhn uses the term “paradigm shift” to describe the change in basic conditions for theory formation in science, such as concept formation, methods of observation and technology used.34 Nowadays, terms such as “theory dynamics” and “theory change” are often used to describe these phenomena. Luca Sciortino35 also mentions Michel Foucault’s ’episteme’36 and the “research program” of Imre Lakatos.37 All these authors thus describe phenomena in connection with change on the basis of changed mindsets and models, ways of thinking and paradigms. These new perspectives lead to a new basis, enable new methods and new technologies and, last but not least, lead to real innovation. Mission statements usually describe a desired goal or an ideal state. They are at home in many areas. They are often used in corporate culture. A mission statement serves as orientation and motivation for the employees of a company, possibly provides information about the product range and activities of a company, can provide a certain framework for the public appearance of a company and even influences the market value of a company. Kühl distinguishes between three sides of a company:38 the so-called “show side”, the “formal side” and the “informal side”. The front side shows the external image, the facade of the company. The so-called formal side forms the official set of rules for all employees. The informal side describes Kühl as the sum of “... ingrained practices and ways of thinking, deviations from official rules and from cultivated myths, dogmas and fictions.” But there are also certain mindsets or paradigms for technical or social developments. They are often closely related to current research and development and formulate certain utopias in these fields. They are ideal models or wishful thinking about future possibilities of technology and society. These models and utopias are often the initial impetus, also for new research projects. Some of the guiding paradigms for digital transformation date back to the 1990s. With “ubiquitous computing” Mark Weiser described an omnipresent mobile use of spatial information accessible to everyone, without visible interfaces and end devices.39 Neil Gross expected “that in the future spontaneous computer networks will emerge and form a ’giant digital creature’.40 He thus describes the current IoT. “Pervasive Computing” and “Ambient Intelligence” also describe related topics today, but with different orientations.41 Ambient Intelligence deals with intelligent systems embedded in the environment that support the user in his activities. In contrast to purely commercial considerations, however, the focus here is often on social and procedural issues. Smart Cities” and “Smart Homes” also belong to these thematic fields. The most important features of ubiquitous computing are the disappearance of hardware and user interfaces, the adaptivity and self-organization of the digital system, automatic context perception, ubiquitous availability of information, and global and local connectivity. I fondly remember a photo of Archigram from the sixties. It shows a telephone lying in a tree trunk, somewhere out in nature. Technical and social utopias are always intertwined. Social models are not always easy to grasp. Tanner describes among other things the concept of “common sense”.42 He understands this to mean social rules and behavior, identity-forming myths and stories, the knowledge of things that “one” does or does not do. “Common sense is the ability to think logically without using specialized or advanced knowledge.”42 The digital transformation in a pluralistic society therefore does not only know one single mindset or paradigm. Companies have their own mission statements, research and development work according to their own ways of thinking and standards, and the population pursues its own individualized life and work ideas, as sometimes, among other things, a sustainable lifestyle. Nevertheless, these mission statements remain powerful drivers for our everyday life.\n\nTable 8 summarizes all clarified concepts and terms from the former literature examination. We can state four stakeholders (politics, research and development, economy and civil society) such as mentioned in Table 1. Sustainability shows up in three different correlations, such as in Table 7. First, it can be a “thought style” for all four stakeholders (P1, R1, E1, S1) (“a new business and corporate culture” in Table 2, “sustainability” and “individualization of work and life models” in Table 3. Second, it can include political actions for climate protection (P2). Third, sustainability can be related to the indicators of primary energy consumption (I1) and the ratio of sustainable processes and technologies (I5) to the total amount of digital processes and technologies (I4) as mentioned in Table 7. These indicators can be completed by the degree of automation (I2) and the digitization speed (I3) as mentioned in Table 5. So we are able to define a group of five indicators finally. The terms of infrastructure investment (P3) (“extension of the digital infrastructure”), financing and coordination (P4) (“governance”, “low interest rate policy”) and prevention of data monopolies (P5) (“data ethics” or “avoidance of data monopolies and better data protection” or “IT security”) were mentioned in Tables 2, 5, 6 and 7. The terms of “demographic change” and “urbanization” as found in Tables 3 and 6 can be expressed in population figures. So both are summarized in the concept of population (S2). Digital literacy (S3) and education (P6) were added as social and political concepts, such as mentioned in Table 2 (“greater digital literacy”), Table 4 (“digital literacy” and “qualified employees training”) and Table 5 (“human capital”). Research and Development (R2) was introduced to reflect the terms of “digitization and technology” and terms of technological development and trends such as in Tables 6 and 7. Product and process quality (E2) was inserted to reflect the terms of “globalization”, “specialization”, “extension of the own portfolio”, “product and process quality” and “efficiency and process optimization” such as in Tables 3, 4 and 6. Digital Usage (S4), affordability (S5), network access (S6), capacity (S7) were adopted directly from Table 5. Mobility (S8) was also listed as in Tables 3 and 7.\n\n\nMethods\n\nAs mentioned in the introduction, FCMs are usually used to investigate complex systems. They consist of a network of concepts and weighted interconnections. The technique of FCMs is often deployed to reveal a dynamic system’s behaviour, describing how the system could evolve in time through causal relationships and for the evaluation of alternatives. Therefore this approach is considered useful in the context of scenario planning and decision making. For example, “Integrated World System Modelling” or an “Integrated Global System Model” is particularly suitable for mapping complex processes using FCMs. The concept of FCMs has been introduced by Bart Kosko in 1986.43 He suggested their use to those knowledge domains that involve an high degree of uncertainty. He extended the work of Axelrod, who found the technique of FCMs representing a natural extension of cognitive maps by embedding to them the use of Fuzzy Logic.44 He also introduced cognitive maps in the context of decision making for the representation of social scientific knowledge and decision making processes in the field of social and political systems. Mention has to be made to the fact that the cognitive mapping approach to decision making uses elements from other fields, such as psychology and graph theory. Since then, FCMs have proven to be a useful method for the systematic collection of knowledge and for the graphical representation of causal relationships and are established in many “hard” and “soft” sciences. Caselles names several research groups that are currently working on global models to simulate the consequences of scenarios and intervention strategies in the world system.45 As prominent examples he mentions the “Regional Earth System” of the Earth System Science Interdisciplinary Centre at the University of Maryland, the “Millennium Project” of the World Bank, the “Integrated Global System Model” of MIT and the “Australian Stocks and Flows Framework (ASFF)”. Bottero et al. for example, successfully use FCMs to model urban resilience dynamics and to support scenario planning and strategic decision making.46 The procedure follows the conventional method of a simulation. Cloud describes the usual professional development of model-building and simulation proceeds in three stages:47 Theoretical basis and monitoring (observation), problem and solution attempts, criticism and elimination of possible errors and mistakes. In the first step you are confronted with a kind of “source system”. This system is real and can be observed or monitored experimentally. Let us assume this is the process of digital transformation in architecture and urban planning. For monitoring and data gathering usually expert interviews, expert opinions or literature reviews are used. Also the delphi method or similar methods can be applied in this initial stage. From there we can derive our input values. The second step is building an abstract model from that source system (idealization and abstraction). Usually only one specific aspect is modeled to clarify one specific question. From the abstract model we can derive an applicable or operable model, for example in a computer simulation. Now we could run an iterative process to test the model and to generate certain scenarios. The output values can be validated against the source system, errors and mistakes can be eliminated and we can start the process again from the beginning. From there we can start an optimization loop. Simulations usually are conducted within a large parameter space to simulate many possible situations. To optimize the target size, the input variables must be varied. This can be done by try-and-error, but this takes a lot of time and effort. Therefore, in the last years, evolutionary optimization methods (e.g. data farming) have been developed to reach the whole realm of a simulation’s test bed. These solutions usually are performed in iterative steps to find an optimal solution. Usually this is done by High Performance Computer (HPC) environments. The results can be depicted in scenarios. The procedure can be repeated until an optimized state is reached. Today, independent learning algorithms help to optimize various system-internal parameters, such as the weighting of correlations. Different learning paradigms and software packages can be distinguished. Felix et al.48 name seven software applications available today: FCM Modeler, FCM Designer, FCM Tool, JFCM, Mental Modeler, ISEMK and FCM Expert. Because of the participatory, web-based solution and because of the ease of use without programming knowledge, this paper uses the Mental Modeler software, developed by Gray et al.16\n\n\n\n• Concepts or components: C1, C2, ... Cn are the system-constituting variables;\n\n• State vector: A = (a1, a2, ... an) where ai is the initial state and the value of the general term Ci. The values assigned to the terms are usually in the range [0;1];\n\n• Directed edges: they symbolize the causality between the terms C1, C2 and are represented as arrows or double arrows;\n\n• Adjacency matrix: E = {eij}, where eij is the weight (w) of the directed edge CiCj. The values assigned to each relation are in the range [−1;1]. The value 0 means, that there is no causal relation between the terms Ci and Cj.\n\nFCMs can be described by different kinds of representation: On the one hand by a graphical representation, the concepts or components and the directed edges. On the other hand by a mathematical representation, which consists of a table mentioning all state vectors in an adjacency matrix. Figure 2 shows a graphical representation of FCM. Table 9 shows the corresponding adjacency matrix.\n\nOne of the great minds of simulation and statistics, George Edward Pelham Box used to say: “All models are wrong, but some of them are useful.”49 This paper presents one of the first applications of FCMs in the context of digital transformation. It aims to design a fuzzy but useful general framework and proof of concept for future inquiry. In this paper terms and concepts for model building are collected from a literature review. Using this “fuzzy” method it is also common for experts to define the correlations in the model. Thus the correlations and causalities correspond to reasonable assumptions by the author. For simulation the Mental Modeler software is used, developed by Gray et al.16 FCMs can describe and simulate the dynamic behavior of systems. Simulating the system behaviour is usually based on mathematical operations and takes place in iterative steps. Unfortunately, the Mental Modeler software is very limited in this respect and does also not allow for the use of dedicated learning algorithms. However, scenario building is very simple, user-friendly and allows for a quick visualization of results. Therefore, this paper only uses the given possibilities of scenario building in the Mental Modeler Software and does not include an additional, algorithmically supported simulation in iterative steps.\n\nThis paper develops an open experimental testbed for sustainable and innovative environments (ETSIE) as a general framework using FCMs. It highlights directions for future inquiry, but it is not expected to collect extensive empirical data. The ETSIE model is developed in the spirit of Open Innovation. The model itself and all data are openly available to all interested people for review and further research. From the current literature review it can be observed that the process of digital transformation is often seen in a predominantely growth-oriented economic sense. During the current pandemic it can be observed as well, that sustainability is not necessarily written in capital letters. Developing innovative and sustainable social and economic models is currently not the first priority.\n\nTherefore three different digital transformation scenarios are designed. A traditional growth-oriented scenario, a COVID-19 scenario and an innovative and sustainable COVID-19 scenario are modeled and tested. All three scenarios have the same number of components, connections and the same driver components. Only the initial state vectors are different and the internal correlations are weighted differently. This allows for comparing all three scenarios on an equal basis. The following three scenarios are illustrated:\n\n\n\n• Traditional, growth-oriented scenario\n\n• COVID-19 scenario (natural disaster)\n\n• Innovative and sustainable COVID 19 scenario\n\nFive indicators provide information on the quality of the different scenarios:\n\n\n\n• Primary energy consumption (I1)\n\n• Degree of automation (I2)\n\n• Digitization speed (I3)\n\n• Total number of existing digital processes and technologies (I4)\n\n• Number of sustainable digital processes and technologies (I5)\n\nInside the given Mental Modeler software, platform data is calculated by the capabilities of the software. For setting the state vectors the software only allows values between -1 and +1. State vectors are set by a reasonable assumption by the author to get significant results. One single scenario is generated at one calculation time. Simulation results are taken directly from the given software. No further iterations, no evolutionary optimization methods or learning algorithms are used. No other than the mentioned scenarios are generated. The three scenarios then subsequently are compared to each other to see, if the ETSIE model delivers useful and significant results and is able to be a proof of concept.\n\nThe following four different groups of people can be identified as stakeholders for the ETSIE model:\n\n\n\n• (P) Politics (cities, counties and municipalities - federal, state and local)\n\n• (R) Research and development\n\n• (E) Economy (companies, architects and urban planners)\n\n• (S) Civil society (population and employees)\n\nThe concepts from Table 8 are directly transferred to Table 10. This table shows the concepts of the ETSIE model with a detailed description. All concepts are assigned to the different stakeholders: politics (Pn), research and development (Rn), economy (En), civil society (Sn) and indicators (In).\n\nIn the following, three alternative scenarios, including the COVID-19 scenario, are depicted using FCMs in the ETSIE model and evaluated according to the five basic indicators mentioned above. Table 11 shows a description of all three alternative scenarios:\n\n\n\n• Scenario 1 - Traditional, growth-oriented scenario\n\n• Scenario 2 - COVID-19 scenario (natural disaster)\n\n• Scenario 3 - Innovative and sustainable COVID-19 scenario\n\nAll three scenarios are built from the same number of concepts (or components) and connections. They all have the same density and the same number of connections per component. All three models have exactly the same complexity score and show six driver components as listed below:\n\n• P1 - Political Thought Style\n\n• R1 - Research Paradigm\n\n• E1 - Business and Corporate Culture)\n\n• S1 - Public Thought Style\n\n• S2 - Population\n\n• S5 - Affordability\n\nThis structural design, the general form of the models, remains the same in all three scenarios. Only the weights of the connections between the nodes (or components) change. This is intended to make it easier and clearer to recognize deviating results and behaviour of the individual models.\n\n\nResults\n\nFigure 3 shows a graphical representation of the first scenario, the traditional growth-oriented scenario. It is characterized by following parameters: The state promotes infrastructure development, research and education. There is no state support for the economy, mobility is not encouraged, sustainable technologies are not promoted, and data monopolies are not avoided. There are no additional government measures for climate protection. The economy additionally supports education, infrastructure extension and the degree of automation. They invest in research for product and process quality, but do not promote sustainable technologies decisively. Research develops new technologies without promoting sustainability. The population is growing moderately and continues to pursue consumption-oriented and individual life and work models, without a significant sustainable share. As a result, government expenses (+0.24) and expenditure on infrastructure extension (+0.46) are increasing. Data monopolies are not avoided (0). Climate protection measures are decreasing (-0.24) and the number of digital processes and technologies is increasing (+0.65), while the share of sustainable technologies is decreasing (-0.7). The digitization speed is increasing (+0.66), as well as the primary energy consumption (+0.72) and the degree of automation (+0.22). There is a higher digital literacy (+0.45), better network access (+0.43), higher capacities (+0.23) and more digital usage (+0.44) and a corresponding higher mobility (+0.46). Corresponding to an increasing product and process quality (+0.44), education (+0.46) and research (+0.44) grow. Table 15 shows the adjacency matrix and the state vectors in scenario 1. The initial state vectors are depicted in white colour on grey ground. Figure 4 shows a graphical depiction of the results of scenario 1 in a bar diagram.\n\nTraditional growth-oriented scenario (Screenshot from Mental Modeler Software).\n\nTraditional growth-oriented scenario (Screenshot from Mental Modeler Software).\n\nFigure 5 shows a graphical representation of the second scenario, the COVID-19 scenario (natural disaster). It is characterized by following parameters: The state is promoting the extension of infrastructure and is also investing more in research. Education and digital literacy receive additional support. There is temporary state support for the economy, mobility is not promoted, sustainable technologies are not promoted, data monopolies are not avoided. There are no additional government measures for climate protection. Companies provide additional support for digital education, infrastructure extension and automation. They also invest in research for product and process quality, but without providing dedicated funding for climate protection or sustainable technologies. Research is increasingly developing new technologies without promoting sustainability. The population is growing moderately, but continues to pursue consumption-oriented and individualized models of living and working, without a significant sustainable share.\n\nCOVID-19 scenario (Screenshot from Mental Modeler Software).\n\nGovernment expenses (+0.36) and expenditures for infrastructure extension (+0.55) are increasing. Data monopolies are not avoided (0). Climate protection measures are decreasing (−0.24). The number of digital processes and technologies is increasing (+0.82), while the share of sustainable technologies is declining (−0.87). The speed of digitization (+0.79), primary energy consumption (+0.79) and the degree of automation (+0.35) are increasing. Digital literacy is increasing (+0.69), there is better network access (+0.66), higher capacities (+0.27) and more digital usage (+0.71). Individual mobility (+0.46) and product and process quality (+0.72) increase. Education (+0.64) and research (+0.79) both grow. Table 16 shows the adjacency matrix and the state vectors in scenario 2. The initial state vectors are depicted in white colour on grey ground. Figure 6 shows a graphical depiction of the results of scenario 2 in a bar diagram.\n\nCOVID-19 scenario (Screenshot from Mental Modeler Software).\n\nFigure 7 shows a graphical representation of the third scenario, the innovative and sustainable COVID-19 scenario. It is characterized by following parameters: There are additional government measures for climate protection. The state promotes the extension of infrastructure and avoids data monopolies. It invests in education, digital literacy and research. There is less state support for the economy. Sustainable mobility is promoted through urban structural measures (e.g. Göderitz et al. “Die gegliederte und aufgelockerte Stadt”50). Individual traffic is reduced. Sustainable technologies are promoted in a targeted manner. The companies support climate protection, education and infrastructure development. The degree of automation is oriented towards a socially sustainable development. The companies invest in research, product and process quality and in sustainable technologies. The population is growing moderately and pursues predominantly socially sustainable and cooperative living and working models, with a high sustainable proportion.\n\nInnovative and sustainable COVID-19 scenario (Screenshot from Mental Modeler Software).\n\nGovernment expenses (+0.36) and expenditure on infrastructure extension (+0.46) are increasing. Additional measures for climate protection (+0.46) and against data monopolies are taken (+0.24). The share of sustainable technologies (+0.96) is increasing, while the total number of digital processes and technologies is being reduced (-0.84). The digitization speed increases (+0.78) while primary energy consumption decreases (-0.76). The degree of automation decreases (-0.36). Digital literacy is increasing (+0.69), there is better network access (+0.64), higher capacities (+0.23) and more digital usage (+0.71). Individual mobility decreases (-0.18). Product and process quality (+0.75), education (+0.64) and research (+0.89) all three grow. Table 17 shows the adjacency matrix and the state vectors in scenario 3. The initial state vectors are depicted in white colour on grey ground. Figure 8 shows a graphical depiction of the results of scenario 3 in a bar diagram.\n\nInnovative and sustainable COVID-19 scenario (Screenshot from Mental Modeler Software).\n\nThe different state vectors of the three alternative scenarios are shown in Table 12. The COVID-19 scenario 2 shows higher state vectors compared to scenario 1. These increased state vectors are applied to illustrate the higher investments, the increased expenditure in opposition to a traditional, growth-oriented scenario 1. Scenario 3 also shows these increased state vectors in many areas, but is supplemented by moderate measures for climate protection. Climate protection will be promoted, general infrastructure expenditure is reduced, data monopolies are avoided and mobility is reduced.\n\nThe results of the scenarios 1 and 2 are similar to a high degree. In both scenarios, the effort for climate protection is reduced to the same extent. The share of sustainable digital processes and technologies decreases in scenario 2 even more. Mobility remains the same, all other factors increase in scenario 2 due to the higher initial state vectors. Table 13 shows the results of the simulation, related to the different scenarios.\n\nScenario 3 differs from the first two scenarios. However, its structure is completely different. Additional measures for climate protection are taken. Conventional government expenses remain at the level of scenario 2. Expenditure on infrastructure returns to the level of scenario 1. Education remains at the same high level. Research and development grow much more strongly than in the first two scenarios. The digitization speed declines only slightly and can almost be maintained at the level of scenario 2. The same applies to network access. Digital usage remains the same. Digital literacy is increased compared to scenario 1 and corresponds to the level of scenario 2. The degree of automation is reduced. Mobility and total energy consumption will decrease. The network capacity can be maintained at the level of scenario 1. The share of sustainable digital processes and technologies can be increased significantly while the total number of digital processes and technologies decreases. Product and process quality increase.\n\nThese results speak for a reachable, innovative and research-driven, sustainable increase in efficiency in scenario 3. With almost the same effort as in scenario 2, a real social and sustainable trend reversal seems possible.\n\nThe network parameters in all three scenarios are completely identical, as shown in Figures 9, 10 and 11. All figures and tables shown in this paper you can easily access in the original version through the provided XML-files in the supplementary materials. They all have the same number of components and connections. They have the same density and the same number of connections per component. All three models have exactly the same complexity score and the same six driver components. Sorting the nodes according to their centrality, you can see the following, as shown in Table 14: All three scenarios differ from each other in the order and evaluation of the centrality parameters.\n\nNetwork parameters and centrality (Screenshot from Mental Modeler Software).\n\nNetwork parameters and centrality (Screenshot from Mental Modeler Software).\n\nNetwork parameters and centrality (Screenshot from Mental Modeler Software).\n\nR2 (Research and Development) has the highest centrality rating in all three scenarios. The centrality of research and development also increases, in scenarios 1 - 3. In scenario 1 (traditional, growth-oriented scenario), the digitization speed is in second place, on a par with product and process quality. Both followed by the share of sustainable processes and technologies. The total number of digital processes and technologies moves up to the fifth place.\n\nInterestingly, in scenario 2 (COVID-19 scenario), product and process quality moves up to second place. Third place is shared by the digitization speed and digital literacy. The order has clearly changed compared to the first scenario. The share of sustainable processes and technologies is in fifth place.\n\nIn scenario 3 (innovative and sustainable COVID-19 scenario), product and process quality retains second place. However, the third place is now clearly followed by the share of sustainable processes and technologies. The digitization speed and digital literacy together fall back to the fourth place.\n\n\nDiscussion and Conclusions\n\nAccording to the results of this study, a sustainable and social turnaround is possible. It is described how a reduction of primary energy consumption and a simultaneous increase in the share of sustainable digital processes and technologies can be achieved with the same or a slightly higher effort. The order of the concepts according to network centrality shows much of what we can already observe in reality today. In all three scenarios, research drives the development of new technologies and is at the center of the digital transformation. All three scenarios have different initial state vectors and digitization speeds. In the traditional and growth-oriented scenario 1, the focus is on digitization speed and on the quality of economic processes and products. The share of sustainable processes and technologies comes after these two factors. This scenario best reflects the well-known and widespread efficiency-oriented and fordist production factors of performance, quality and acceleration. In COVID-19 scenario 2, the quality of products and processes takes second place, followed by digitization speed. With higher initial state vectors and a higher effort the product and process quality increases. The digitization speed still plays an important role, but loses centrality. It is shown that there is a factual connection to a general “slowdown” in the course of the current pandemic. Interestingly, digital literacy now appears to be more prominent. The share of sustainable processes is slipping further behind. The simulation results show, that in times of COVID-19, sustainability is not necessarily written in capital letters, and we can observe this as well in reality. In scenario 3, the innovative and sustainable COVID-19 scenario, research is also at the top of the list, followed by product and process quality. However, the share of sustainable processes and technologies now takes on a central position. The digitization speed and digital literacy are moving further back, but are still in the focus of our interests. Taking into account digital education and a moderate digitization speed, further improvement of our products and processes can lead to sustainable quality and real innovation. For architecture and urban planning these results are very important. Innovative digital transformation will only be possible by a general, integrative and coordinated change of “thought styles” of all stakeholders involved. Innovative sustainability can be reached through more intensive and target-oriented research and development from both sides, economy as well as science. Research and development will be the basis for really innovative and sustainable products and services. Therefore we will need a reliable classification of sustainable technologies. Additionally an adequate digital literacy and responsible public handling of resources will be needed. Mobility can be optimized through appropriate urban structure planning. It will not only be the question of a new category of vehicles. We all will have to review our lifestyle. Urban planning will have to intensively review the city structures for innovative and sustainable principles and generate ideas for sustainable city quarters of the future. Where do we want to live, where do we work, do we really have to travel that much? And we all will see processes slowing down to a socially compatible, sustainable and human level. Technological development already fulfils many future scenarios today. Even if we are not far away from the adaptivity and self-organisation of digital systems, there is still a long way to go for automatic context perception and comprehensive standards. It would also be desirable to have a solid, worldwide non-profit organization in order to make high-quality data openly available. For weather data, the World Meteorological Organization of the UN has been in existence for 70 years. Currently, there are also few standardized assessments of digital processes and technologies. Often no clear distinction is made between conventional and sustainable digital technologies. Often the mutual basics are missing. A catalog of innovative and sustainable social processes and technologies for targeted promotion and financing would be desirable. The degree of automation and social compatibility of innovative processes and technologies should also play a key role in this context. A central organization at global or european level could certainly make a significant contribution to this. For future work there are many interesting perspectives. The ETSIE model provided a first proof of concept comparing three scenarios of digital transformation using a relatively simple software basis. Of course the figures can be improved to illustrate the advantages of the innovative and sustainable model. In the next steps the number of scenarios could be extended using another software platform or a combination of advanced software to reach the whole realm of the experimental simulation’s testbed. Generating more scenarios. using learning algorithms and evolutionary optimization and evaluation will lead to refinement and general improvement of the model and should provide new and more detailed information on the general process of digital transformation in architecture andurban planning. So, will COVID-19 accelerate digitization? To a certain extent yes. This study has shown that COVID-19 can be an accelerator of digitization, but is no guarantee for sustainable and high quality innovation. Similar to a natural disaster, COVID-19 can be presented as just one of many possible digitization scenarios. The investments and all the stakeholders decision will show which direction we want to take. Whether we want to continue to pursue primarily commercial interests, or whether we see the sustainable benefits of a solidary community in a healthy and functioning, really innovative environment. It will not be enough to digitize the existing growth-driven economy. A fundamental and innovative change is necessary to avert a dramatic climate catastrophe and to solve our social problems. This change must be supported by all stakeholders in the same way and can be implemented with nearly the same or a moderate higher effort. The current crisis is an opportunity for new ideas, for the meaningful restructuring of creative and productive processes. But this does not only include investment in infrastructure and digital literacy. How we want to keep it, that way is up to us to decide. The question remains - in which world do we want to live?\n\n\nData availability\n\nmediaTUM: COVID-19 and Digital Transformation - Developing an Open Experimental Testbed for Sustainable and Innovative Environments (ETSIE) using Fuzzy Cognitive Maps, https://doi.org/10.14459/2020md1601121.51\n\nThis project contains the following underlying data:\n\n• Traditional growth input data for Mental Modeler software\n\n• Normal COVID-19 input data for Mental Modeler software\n\n• Sustainable COVID-19 input data for Mental Modeler software\n\nData are available under the terms of the Creative Commons Attribution NonCommercial 4.0 International license (CC-BY-NC 4.0).", "appendix": "Acknowledgements\n\nThe author would like to thank Theresa Ramisch from Baumeister Magazine at Georg Media GmbH. in Munich, who asked me to have a closer look at this interesting topic. My thanks also go to my partner Cornelia for her patience and support developing this small research project and her valuable advice. Last but not least, my sincere gratitude goes to Gudrun Klinker at TUM, who kindly made this publication possible.\n\nA previous version of this article can be found on arXiv: http://export.arxiv.org/abs/2101.07509.\n\n\nReferences\n\nCoronavirus COVID-19 Global Cases by the Center for Systems Science and Engineering (CSSE) at Johns Hopkins University (JHU) in Baltimore, Maryland / USA.\n\n“Die Welt und die Gesellschaft in Deutschland wird sich verändern”.Deutschlandfunk; 2020.\n\nKMK-Präsidentin erwartet Schub für Digitalisierung an Schulen.Deutschlandfunk; 2020.\n\nSchmidt P, Ebner S: Petition gegen den Fernunterricht, Münchner Merkur.2020 Nr. 71, 25-03-2020, p. 16.\n\nZadeh LA: The roles of fuzzy logic and soft computing in the conception, design, and deployment of intelligence systems. in: Nwana HS, Azarmi N (eds) Software agents and soft computing: towards enhancing machine intelligence concepts and applications. Lecture notes in computer science. 1997; vol. 1198, pp 83–90.\n\nZadeh LA: What is Soft Computing. Soft Comput. 1997; 1: 1–2.\n\nBonissone P: Soft computing: the convergence of emerging reasoning technologies. Soft Comput. 1997; 1: 6–18.\n\nJain L: Soft computing techniques in knowledge-based intelligent engineering systems: approaches and applications. Studies in fuzziness and soft computing. Berlin Heidelberg New York: Springer; 1997; vol. 10.\n\nPapageorgiou EI, Salmeron JL: A review of fuzzy cognitive maps research during the last decade. IEEE Trans.Fuzzy Syst. 2013; 21(1): 69–79. Publisher Full Text\n\nÖzesmi U, Özesmi S: A participatory approach to ecosystem conservation: fuzzy cognitive maps and stakeholder group analysis in Uluabat Lake, Turkey. Environ. Manage. 2003; 31(4): 518–531. PubMed Abstract \| Publisher Full Text\n\nÖzesmi U, Özesmi SL: Ecological models based on people knowledge: A multy – step fuzzy cognitive mapping approach. Ecol. Model. 2004; 176: 43–64. Publisher Full Text\n\nMisthos LS, Messaris G, Damigos D, et al.: Exploring the perceived intrusion of mining into the landscape using the fuzzy cognitive mapping approach. Ecol. Eng. 2017; 101: 60–74. Publisher Full Text\n\nJetter A: Fuzzy cognitive maps for engineering and technology management: what works in practice? In: PICMET Proceedings. 2006.\n\nJetter A, Schweinfort W: Building scenarios with fuzzy cognitive maps: an exploratory study of solar energy. Futures. 2010; 43: 52–66. Publisher Full Text\n\nPapageorgiou EI, Groumpos PP: A weight adaptation method for fuzzy cognitive map learning. Soft Comput. 2005; 9: 846–857. Publisher Full Text\n\nGray SA, Gray S, Cox LJ, et al.: Mental Modeler: A Fuzzy-Logic Cognitive Mapping Modeling Tool for Adaptive Environmental Management. In: Proceedings of 46th Hawaii International Conference on System Sciences. Maui, HI: Wailea; 2013; pp. 965–973.\n\nHorx M: Die Zukunft nach Corona - Wie eine Krise unsere Gesellschaft, unser Denken und unser Handeln verändert.Berlin: Econ Verlag; 2020.\n\nWidmann A: Ist die Corona-Pandemie eine Naturkatastrophe? Der Standard.2020, April 20th.\n\nDetting D: Zukunftsszenarien nach Corona - Gesundheit wird nicht mehr nur eine individuelle Angelegenheit sein, Der Tagesspiegel.2020, April 5th.\n\nvon der Gracht H: Die Zukunft nach Corona, KPMG Klardenker (eds.) 2020, July 2nd.\n\nThierstein A: Digitale Transformation im urbanen Raum, Stadt Bauwelt. Nr. 219 / 19.2018 – Digitale Stadt. 2018; pp. 32–35.\n\nGoger G, Piskernik M; Urban, Harald. Studie: Potenziale der Digitalisierung im Bauwesen. Bundesministerium für Verkehr. Vienna / Austria: Innovation und Technologie and Wirtschaftskammer Österreich, Geschäftsstelle Bau; 2018.\n\nRoland Berger Holding GmbH: (eds.). Wirtschaftliche Auswirkungen des Coronavirus - Teil 2. Roland Berger. 2020, March 26th; München.\n\nInternet Economy Foundation (IE.F); Roland Berger Holding GmbH: (eds.) Deutschland digital - Sieben Schritte in die Zukunft.(undated).\n\nKatz R, Koutroumpis P, Callorda F: Using a digitization index to measure the economic and social impact of digital agendas. info. Vol. 2014; 16 No. 1, pp. 32–44. Publisher Full Text\n\nRoland Berger Holding GmbH: (eds.). Digitales Arbeiten in Zeiten von COVID-19. Roland Berger. 2020, April 2nd; München.\n\nSchüller K, Förster A: Digital Literacy für die Stadt. Informationen zur Raumentwicklung. 2017; 1, p. 108–121.\n\nSimondon Gilbert: Die Existenzweise technischer Objekte. diaphanes. Zürich.\n\nBaumanns T, et al.: Bauwirtschaft im Wandel, Trends und Potenziale bis 2020.München: Studie Roland Berger GmbH. und UniCredit Bank AG; 2016.\n\nBundeskanzleramt und Bundesministerium für Wissenschaft, Forschung und Wirtschaft: (eds.). Digital Roadmap Austria.Vienna, Austria. 2016, December.\n\nBRZ Deutschland GmbH: (eds.). IT-Trends in der Baubranche 2016 - Status quo und Perspektiven. Nürnberg. 2016.\n\nHacking I: ‘Style’ for historians and philosophers. Studies in History and Philosophy of Science. March 1992; Part A, Volume 23, Issue 1, pp. 1–20.\n\nFleck L: Entstehung und Entwicklung einer wissenschaftlichen Tatsache - Einführung in die Lehre vom Denkstil und Denkkollektiv.1980. Suhrkamp Verlag; Frankfurt.\n\nKuhn TS: The Structure of Scientific Revolutions.Chicago: University of Chicago Press; 1962.\n\nSciortino L: On Ian Hacking’s Notion of Style of Reasoning. Erkenntnis - An International Journal of Scientific Philosophy. 2017; 82, pp. 243–264.\n\nFoucault M: The order of things.1994. Vintage Books; New York.\n\nLakatos I: The methodology of scientific research programmes. Cambridge Univ. Press; 1978.\n\nKühl S: Leitbilder erarbeiten, Eine kurze organisationstheoretisch informierte Handreichung, Universität Bielefeld.2017; Springer Fachmedien Wiesbaden.\n\nWeiser M: The Computer of the 21st Century. Scientific American. 1991; 265 (3).\n\nGross N: The Earth Will Don An Electronic Skin, Bloomberg Business.1999 August 30th.\n\nWiegerling K: Ubiquitous Computing als konkrete Utopie. In: Grimm P, Capurro R (eds.). Informations- und Kommunikationsutopien. Stuttgart: Franz Steiner Verlag; 2008.\n\nTanner K: Common Sense: Get It, Use It, and Teach It in the Workplace.Apress Business; 2013.\n\nKosko B: Fuzzy cognitive maps. In: International Journal of Man-Machine Studies 1986; Volume 24, Issue 1, Pages 65–75. 0020-7373.\n\nAxelrod R: Structure of Decision: The Cognitive Maps of Political Elites.Princeton: Princeton University Press; 1976.\n\nCaselles A: An application of fuzzy cognitive maps to improve well-being, sustainability and the globalization process. In: Systems Research and Behavioral Science. November 2013; Vol.30(6), pp. 646–660.\n\nBottero M, Datola G, Monaco R: Exploring the Resilience of Urban Systems Using Fuzzy Cognitive Maps. In: Gervasi O, et al. (eds) Computational Science and Its Applications – ICCSA. 2017, 2017, Lecture Notes in Computer Science, vol 10406.Springer; Cham.\n\nCloud DJ: Applied Modeling and Simulation. New York: McGraw-Hill; 1998.\n\nFelix G, Nápoles G, Falcon R, et al.: A review on methods and software for fuzzy cognitive maps. Artif Intell Rev. 2019; 52: 1707–1737.\n\nBox GEP: Empirical Model-Building and Response Surfaces.1987. Wiley.\n\nGöderitz Johannes, Rainer Roland, Hoffmann Hubert: Die gegliederte und aufgelockerte Stadt.Tübingen; 1957.\n\nHöhl W: COVID-19 and Digital Transformation - Developing an Open Experimental Testbed for Sustainable and Innovative Environments (ETSIE) using Fuzzy Cognitive Maps. mediaTUM [dataset]. Publisher Full Text" }	[ { "id": "86940", "date": "14 Jun 2021", "name": "Peter Zeile", "expertise": [ "Reviewer Expertise Urban Planning", "Digital Methods in Urban Planning and Architecture" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nF1000Research: COVID-19 and digital transformation: developing an open experimental testbed for sustainable and innovative environments using Fuzzy Cognitive Maps\nContent This paper sketches a new approach using Fuzzy Cognitive Maps (FCMs) to operably maps and simulate digital transformation in architecture and urban planning. Today these processes are poorly understood. The paper introduces an experimental testbed in which economics, sustainability and social impacts were merged and develops three different digital transformation scenarios using FCMs\nGeneral Comments Is the ETSIE model your own developed model? If yes, please clarify it in the introduction and the abstract.\nFor a better understanding, I would shorten the introduction or insert the section on FCMs as a separate chapter and structure it a little better with paragraphs. As we \"jump\" from the social component very quickly into the matter of FCMs, go into detail here with the advantages and disadvantages already, which may be easier to understand when viewed separately.\nPerhaps it would also be helpful to mention before the literature evaluation that the evaluation serves to \"feed\" the software so that the inserted tables are easier to understand at this point.\nOverall, the contribution \"on the road\" loses focus, since the actual question is whether we can better assess the consequences of digitalisation in urban planning and architecture. The topic is only taken up again in the discussion.\nFurthermore, as a reader who is not completely familiar with the matrix, one feels somewhat lost along the way; the individual steps are usually explained logically, but the big picture is sometimes not visible. Perhaps an exemplary workflow or flow chart would be useful, what data comes in where, which transformations and extracts are integrated into the Mental Modeler software, and what does that mean in the results if I am +0.50 \"better\".\nWhat the contribution does, however, is to summarise the framework conditions in the scenarios in such a way that the respective interactions can be (better?) assessed. These results are a good basis for discussing guiding principles, visions and goals at the political level.\nOf course, the question is always how much \"trust\" decision-makers have in such software that the consequences will occur when I turn screw X, (e.g. that the degree of digitalisation will decrease). and I think this is exactly where the article could sharpen the process or the \"how\", as described above with a flow chart.\nIt would also be very helpful to have a few more specific statements on architecture and urban planning.\nIn Details Introduction: Please add that they are in the context of the German digital transformation, the ministers are probably not familiar to the international audience.\n\"A study by BRZ Deutschland GmbH. names the following six IT trends in the construction industry.\" -> Which ones? See table 7?\nOverall Recommendations Overall, an article with a lot of potentials, but with a little more structure and mapping based on the points mentioned above are still needed.\n\nIs the rationale for developing the new method (or application) clearly explained? Partly\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly", "responses": [] }, { "id": "87003", "date": "21 Jun 2021", "name": "Tamás Molnár", "expertise": [ "Reviewer Expertise Historical and contemporary development of architecture" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper has generally a right structure. The description of the presented methodology is scientifically well based and clearly presented.\nSome small corrections would be needed to improve the paper:\nIt is not the best solution to repeat a certain part of the abstract in the same way in the Introduction. Reformulating this part of the paper can make the introduction a little bit shorter.\n\nThe abbreviation of BIM appears already in the earlier part of the Degree of automation chapter. The full name should be written there instead of the current place.\n\nIt should be clarified what is the Münchener Kreis. At least an English translation should be provided, it would be even better to have a clarifying sentence.\n\nInstead of personal pronouns it is better to use the Author or passive voice e.g. the Author thinks or it is thought. In the Technologies chapter it is written “To my opinion”, in the Thought styles chapter it is written I fondly remember. These sentences should be corrected.\n\nThe order of the tables are mixing up at the end of the paper. Table 16 and 17 should appear next to the text where it is mentioned, practically at the description of the scenarios. It is contradictory that Table 12 comes later than Table 16.\n\nThere are a few spelling mistakes that should be corrected.\nThe overall evaluation of the paper is good and it is surely useful for further research in the challenging times of the current COVID-19 world.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [] }, { "id": "88763", "date": "26 Jul 2021", "name": "Chrysostomos D. Stylios", "expertise": [ "Reviewer Expertise Soft computing", "Fuzzy Cognitive Maps" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nContent This paper proposes the application of Fuzzy Cognitive Maps (FCMs) to operably maps and simulate digital transformation in architecture and urban planning. By using this soft computing approach, the authors concluded that COVID-19 accelerates digitization to some extent.\nGeneral Comments\nThe introduction must give a map of the paper. By the end of the introduction, reader should have a pretty good idea of what they're in for, and where they're going to end up. They should know what sections they're probably going to skip, and which ones they look forward to disagreeing with. So it would be advisable to write a brief organization of the paper.\n\nSecond person pronouns are a good technique for giving instructions but it is not appropriate in academic or scientific writing.\n\nIn page 3, the sentence “But there also other interesting solutions” could have been merged into one with the previous.\n\nAvoid repetition at the paper. Don't restate points you've already made.\n\nUse the first person singular pronoun appropriately, for example, to describe research steps or to state what you will do in a chapter or section. Do not use first person \"I\" to state your opinions or feelings.\n\nTable 2, 3,4, 5 and 6 could be combined in one table with 5 columns (one for each author).\n\nHow the weight matrix was defined? In this paper the determination of the concepts is described extensively. More technical details about the weight matrix of each scenario should be mentioned (The causal interrelationships among concepts were declared using linguistic variables by an expert panel and a defuzzification method?).\n\nThe FCM that have been developed (determination of the weight matrix) by the experts involve their subjectivity. Why was the application of a training algorithm for the weight adjustment not preferred?\nOverall Recommendations There are many research papers that describe FCM development the more detailed explanation and various papers that investigate FCMs application and FCM in similar contexts so it is good to see work in this area and include them as references.\n\nIs the rationale for developing the new method (or application) clearly explained? Partly\n\nIs the description of the method technically sound? Partly\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-264
https://f1000research.com/articles/10-262/v1	01 Apr 21	{ "type": "Systematic Review", "title": "Organochlorine pesticide exposure and risk of prostate cancer development and progression: a systematic review", "authors": [ "Laurent Brureau", "Luc Multigner", "Freddie Hamdy", "Pascal Blanchet", "Richard Bryant", "Luc Multigner", "Freddie Hamdy", "Pascal Blanchet", "Richard Bryant" ], "abstract": "Background: There is an increasing body of evidence linking the exposure of an individual to pesticides such as organochlorine pesticides (OPCs) and an increased risk of developing diseases such as cancer. Exposure to OPCs has been suggested to increase the risk of developing hormone-dependant cancers such as prostate cancer (PCa). However, there is a relative paucity of information about the influence of exposure to these pesticides on the evolution of PCa, including risk of tumour development, progression to metastasis, and disease recurrence following therapy. Methods: We used several databases such as PubMed MEDLINE Database, Web of Science, and Scopus, in order to conduct a systematic review of the available epidemiological data implicating an association between exposure to OCPs and biochemical recurrence (BCR) of PCa. We searched all peer-reviewed articles published up to July 31st 2020. Pre-defined eligibility criteria for the inclusion of studies were that they be original studies, reviews, previous meta-analyses, or case–control or cohort studies. Results: Agent Orange is the most widely-studied OCP in the context of any possible causal role in the recurrence of PCa following radical prostatectomy, or in the progression to advanced disease. Only two studies didn’t demonstrate a significant association between exposure to OCPs and subsequent BCR following radical prostatectomy. Another study identified a significant association between exposure to Oxychlordane and PCB44 and progression to advanced PCa. Conclusion: This review confirmed a relative lack of high-quality evidence regarding this topic. However, the available evidence to date suggests the presence of a potential causal relationship between exposure to OPCs and PCa development and progression.", "keywords": [ "Organochlorine pesticides", "prostate cancer", "biochemical recurrence", "advanced disease" ], "content": "Abbreviations\n\nAO: Agent Orange; BCR: Biochemical Recurrence; OCs: Organochlorines; OCPs: Organochlorine Pesticides; PCa: Prostate Cancer\n\n\nIntroduction\n\nProstate cancer (PCa) is the second most common non-cutaneous malignancy diagnosed among men worldwide, and the most common cancer type detected in men in developed countries1. Several risk factors for the development of PCa have been established, including increasing age, positive family history, and accumulated environmental exposure to several hormones2,3. Some pesticides can influence the hormonal milieu in vivo by functioning to mimic the effect of hormones, regulate enzyme systems involved in hormone metabolism, and affect androgenic stimulation of the prostate gland, potentially leading to increased cellular proliferation and progression to malignancy4–6. Organochlorines (OCs) comprise a large number of pesticides, and these have been used extensively throughout the world for several decades. Whilst their use has been banned or severely restricted in many countries, they remain in use in many areas of the world, and this has the potential to adversely affect the health of individuals in countries where OCs are still in use. OCs are highly-persistent organic pollutants, with a high serum level being reported in several distinct populations7–10. The International Agency of Research on Cancer (IARC) has classified many OCPs as being Class 2B agents, implicating them as being possible carcinogens11. Moreover, a large number of OCPs have been demonstrated to have the potential to disrupt endocrine function12,13, suggesting that exposure to these specific types of pesticides may increase the risk of developing hormone-dependant cancers such as PCa14. Several OCPs including chlordecone, DDE, DDT and Lindane have been implicated as potential independent risk factors for PCa development15–18. However, to date there is a relative lack of information about the impact of exposure to OCPs upon on the development of aggressive metastatic PCa, or influences on PCa disease-free survival, and potential BCR following radical treatment. The aim of this review article is to provide a contemporary update of the epidemiologic evidence implicating exposure to OCPs upon the recurrence of PCa following radical therapy.\n\n\nMethods\n\nWe conducted a systematic review of the available epidemiological data investigating a potential relationship between exposure to OCPs and the development of recurrent PCa following radical therapy. We searched all peer-reviewed articles published up to July 31st 2020. Pre-defined eligibility criteria for the inclusion of studies were that they be original studies, reviews, previous meta-analyses, or case–control or cohort studies. Moreover, it was mandatory that they contain information about association measures, including odds ratios (OR), relative risks (RR), and confidence intervals (CI) in order to facilitate an analysis of possible relationships between exposure to specific OCPs and development of recurrent PCa following treatment. Finally, it was necessary for the included studies to provide sufficient data and be written in English, French, or Spanish. Exclusion criteria included in vitro experimental and mechanistic studies, editorials, or letters, and as such these reports were not included in this review.\n\nThe initial search strategy included PubMed MEDLINE Database, Web of Science, and Scopus, utilising different “key words” to order identify studies investigating potential associations between exposure to OCPs and development of recurrent PCa following treatment (Figure 1).\n\nMeSH controlled vocabulary was utilised, including combinations of the following key words: ‘‘organochlorine pesticides’’, ‘‘exposure’’, ‘‘DDT’’, ‘‘DDE’’, ‘‘hexachlorocyclobenzene’’, ‘‘lindane’’, ‘‘chlordecone’’, ‘‘kepone’’, ‘‘chlordan’’, ‘‘dicofol’’, ‘‘mirex’’, ‘‘dieldrin’’, ‘‘endrine’’, ‘‘aldrine’’, ‘‘PCB’’, ‘‘dioxine’’, ‘‘endosulfan’’, ‘‘heptachlor’’, ‘‘methoxychlor’’, ‘‘toxaphene’’, ‘‘prostate cancer’’, ‘‘biochemical recurrence’’, ‘‘biochemical failure’’, ‘‘prostatic carcinoma’’, ‘‘prostatic neoplasm’’, ‘‘prostatic adenocarcinoma’’, ‘‘case – control studies’’ and ‘‘cohort studies.’’\n\nExtracted domains included sttudy design, demographics, findings\n\n\nResults\n\nAn overview of five available studies investigating a potential relationship between exposure to OCPs and development of recurrent PCa following radical treatment is provided in Table 1. Two studies did not observe any significant relationship between the exposure of American Veterans to Agent Orange (AO) and subsequent BCR following radical prostatectomy19,20. Li et al. reported that exposure to AO significantly increased the Dioxin-TEQ level in blood samples (p < 0.001), but high dioxin-TEQ levels were not associated with an increased risk of subsequent BCR (p=0.23). A study by Ovadia et al. found that men exposed to AO did not have an increased risk of BCR following radical prostatectomy in both a univariate analysis (HR 1.03; 95% CI 0.84 – 1.25; p=0.80) and a multivariate analysis (HR 1.21; 95% CI 0.99–1.49; p=0.07). However, a study by Shah et al. reported a significant positive association between exposure to AO and BCR following radical prostatectomy. In this study of 206 men, those with documented exposure to AO had a significantly increased risk of subsequent BCR following radical prostatectomy (RR 1.55; 95% CI 1.15 – 2.09; p=0.004 when adjusted for clinical characteristics, and RR: 1.47; 95% CI 1.08 – 2.00; p=0.02 when adjusted for clinical plus pathological characteristics)21. Another study by Brureau et al. revealed a significant positive association between exposure to Chlordecone and BCR following radical prostatectomy and no associations for DDE or PCB-135. In this study of 326 men, those with documented exposure to Chlordecone had a significantly increased risk of subsequent BCR following radical prostatectomy (adjusted HR = 2.51; 95% CI: 1.39 – 4.56; for the highest versus lowest quartile of exposure; p trend = 0.002). In addition, sensitivity analysis revealed that Chlordecone exposure was still significantly associated with a risk of BCR after excluding patients with positive surgical margins or prostatectomy ISUP Gleason grade 3 or higher, or advanced pathological stage22.\n\nA report by Koutros et al. suggests that other pesticides, such as Oxychlordane and PCB44, may be implicated in modifying the risk of developing advanced PCa. For example, the development of metastatic PCa was twice as likely among men with a serum concentration of Oxychlordane in the highest quartile when compared against those in the lowest quartile (OR 2.03; 95% CI 1.03 – 4.03; p-trend=0.05). Findings for specific PCB-related chemicals showed a significant inverse association between natural log–transformed lipid-adjusted PCB44 and metastatic PCa (OR 0.74; 95% CI 0.56–0.97; p-trend=0.02)23. All characteristics of OCPs involved in BCR or metastatic PCa are summarised in Table 2.\n\n\nDiscussion\n\nThis systematic review confirms that there is a relative lack of high-quality evidence implicating a potential association between exposure to OCPs and BCR of PCa. However, the available evidence suggests that there may be a potential causal relationship between exposure to OCPs and development and progression of this malignancy. Agent Orange is the most widely-studied OCP in the context of any possible causal role in the recurrence of PCa following radical prostatectomy, or in the progression to advanced disease24. However, only two studies demonstrated a significant association between exposure to OCPs and subsequent BCR following radical prostatectomy. Two pesticides were involved: Chlordecone and Agent Orange21,22. Another study by Koutros et al. identified a significant association between exposure to Oxychlordane and PCB44 and progression to advanced PCa23. However, each of these studies are limited by their inclusion of a relatively small number of cases. Larger prospective clinical studies would be necessary to confirm these potential associations, however it is recognised that such studies would be very difficult to conduct, and are not presently feasible.\n\nThis review highlights the relative lack of evidence on the potential causal role of OCPs in PCa development and progression, despite the observation that a large number of pesticides exist and continue to be in use in many countries worldwide (Table 3). As such, this topic has potential impacts in aspects of global healthcare, and there is widespread public concern regarding pesticide exposure and negative impacts on health29. There is a well-documented causative relationship between exposure to pesticides and increased risk of development of many types of malignancy. It is therefore important to understand in greater detail the potential influence of OCP exposure upon aspects of PCa risk, and to identify the molecular pathways and mechanisms implicit in this increased risk (Figure 2).\n\nT3L = lower tertile 3, T3U = uppertertile3, Q1, Q2, Q3, Q4 = Quartiles, Qu1, Qu2, Qu3, Qu4, Qu5 = Quintiles\n\na Included aldrin, chlordane, DDT, dieldrin, heptachlorandtoxaphene\n\nb Dicofol, dieldrin, dienochlor, endosulfan, heptachlor, lindane, methoxychlor, and toxaphene\n\nSome OCPs, such as PCBs and Chlordecone, have functional properties that disrupt various endocrine pathways, including the synthesis, secretion, transport, and binding of hormonal ligands to their cognate receptors, whilst in addition they may result in the elimination of natural human hormones30. Phthalte pesticides are endocrine disruptor molecules with demonstrable estrogenic effects in breast and PCa cells, and these may also be implicit in the etiology of hormone-independent PCa cancer31. Given that phthalates are estrogen-like substances, they can positively regulate the proliferation of human hormone dependent PCa cells by acting on the crosstalk between TGF-β and oestrogen receptor signaling pathways32. In addition, some studies suggest that estrogen and xenobiotic carcinogens may play an important role in PCa progression via oxidative estrogen metabolism. For example, the CYP1B1 enzyme is involved in the hydroxylation of estrogens, and this reaction is of key relevance to the regulation of estrogen metabolism33. The over-production of estrogen-like E2, or the bioconversion of E2 into genotoxic metabolites such as estradiol-3,4-quinone or 4-hydroxyestradiol by CYP1B1, may lead to the generation of reactive oxygen species which subsequently cause DNA damage and enhance PCa progression34. In support of this hypothesis, Gu et al. observed that men with the CY1B1 rs1056836 CC genotype had an increased risk of PCa recurrence following radical prostatectomy when compared against a combined CG and GG genotype35.\n\n\nConclusion\n\nIn conclusion, this review highlights the relative lack of studies regarding the potential influence of OCPs on the recurrence and progression of PCa following radical therapy. An increased understanding of the pathways and mechanisms through which pesticides may influence the natural history of PCa progression could influence the clinical management of men with this ubiquitous and common malignancy. It is important that the current relatively small body of evidence demonstrating a negative influence of OCPs on PCa risk should be added to in as timely a fashion as possible so that knowledge of this important health topic can increase, with a resultant positive health benefit for a significant number of individuals worldwide.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nFigshare: PRISMA checklist for ‘Organochlorine pesticide exposure and risk of prostate cancer development and progression: a systematic review’, https://doi.org/10.6084/m9.figshare.14245694.v136.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nCenter MM, Jemal A, Lortet-Tieulent J, et al.: International variation in prostate cancer incidence and mortality rates. Eur Urol. 2012; 61(6): 1079–92. PubMed Abstract \| Publisher Full Text\n\nChen C, Weiss NS, Stanczyk FZ, et al.: Endogenous sex hormones and prostate cancer risk: a case-control study nested within the Carotene and Retinol Efficacy Trial. Cancer Epidemiol Biomarkers Prev. 2003; 12(12): 1410–1416. PubMed Abstract\n\nHsing AW: Hormones and prostate cancer: what’s next? Epidemiol Rev. 2001; 23(1): 42–58. PubMed Abstract \| Publisher Full Text\n\nKeller-Byrne JE, Khuder SA, Schaub EA: Meta-analyses of prostate cancer and farming. Am J Ind Med. 1997; 31(5): 580–586. PubMed Abstract \| Publisher Full Text\n\nJanssens JP, Van Hecke E, Geys H, et al.: Pesticides and mortality from hormone-dependent cancers. Eur J Cancer Prev. 2001; 10(5): 459–467. PubMed Abstract \| Publisher Full Text\n\nParent ME, Siemiatycki J: Occupation and prostate cancer. Epidemiol Rev. 2001; 23(1): 138–143. PubMed Abstract \| Publisher Full Text\n\nYang L, Li X, Zhang P, et al.: Concentrations of DDTs and dieldrin in Long Island Sound sediment. J Environ Monit. 2012; 14(3): 878–885. PubMed Abstract \| Publisher Full Text\n\nPark MJ, Lee SK, Yang JY, et al.: Distribution of organochlorines and PCB congeners in Korean human tissues. Arch Pharm Res. 2005; 28(7): 829–838. PubMed Abstract \| Publisher Full Text\n\nJaga K, Dharmani C: Global surveillance of DDT and DDE levels in human tissues. Int J Occup Med Environ Health. 2003; 16(1): 7–20. PubMed Abstract\n\nJakszyn P, Goñi F, Etxeandia A, et al.: Serum levels of organochlorine pesticides in healthy adults from five regions of Spain. Chemosphere. 2009; 76(11): 1518–1524. PubMed Abstract \| Publisher Full Text\n\nIARC, IARC monographs on the evaluation of carcinogenic risks to humans Volume 100F: A review of human carcinogens: chemical agents and related occupations. International Association for Research on Cancer (IARC), Lyon. 2012.\n\nDe Coster S, Van Larebeke N: Endocrine-disrupting chemicals: associated disorders and mechanisms of action. J Environ Pub Health. 2012; 2012: 713696. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcKinlay R, Plant JA, Bell JNB, et al.: Endocrine disrupting pesticides: implications for risk assessment. Environ Int. 2008; 34(2): 168–183. PubMed Abstract \| Publisher Full Text\n\nLandau-Ossondo M, Rabia N, Jos-Pelage J, et al.: Why pesticides could be a common cause of prostate and breast cancers in the French Caribbean Island, Martinique. An overview on key mechanisms of pesticide-induced cancer. Biomed Pharmacother. 2009; 63(6): 383–395. PubMed Abstract \| Publisher Full Text\n\nMills PK, Yang R: Prostate cancer risk in California farm workers. J Occup Environ Med. 2003; 45(3): 249–58. PubMed Abstract \| Publisher Full Text\n\nBand PR, Abanto Z, Bert J, et al.: Prostate Cancer Risk and Exposure to Pesticides in British Columbia Farmers. Prostate. 2011; 71(2): 168–183. PubMed Abstract \| Publisher Full Text\n\nMultigner L, Ndong JR, Giusti A, et al.: Chlordecone Exposure and Risk of Prostate Cancer. J Clin Oncol. 2010; 28(21): 3457–3462. PubMed Abstract \| Publisher Full Text\n\nEmeville E, Giton F, Giusti A, et al.: Persistent organochlorine pollutants with endocrine activity and blood steroid hormone levels in middle-aged men. PLoS One. 2013; 8(6): e66460. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi Q, Lan L, Klaassen Z, et al.: High level of dioxin-TEQ in tissue is associated with Agent Orange exposure but not with biochemical recurrence after radical prostatectomy. Prostate Cancer Prostatic Dis. 2013; 16(4): 376–81. PubMed Abstract \| Publisher Full Text\n\nOvadia AE, Terris MK, Aronson WJ, et al.: Agent Orange and long-term outcomes after radical prostatectomy. Urol Oncol. 2015; 33(7): 329.e1–6. PubMed Abstract \| Publisher Full Text\n\nShah SR, Freedland SJ, Aronson WJ, et al.: Exposure to Agent Orange is a significant predictor of prostate-specific antigen (PSA)-based recurrence and a rapid PSA doubling time after radical prostatectomy. BJU Int. 2009; 103(9): 1168–1172. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrureau L, Emeville E, Helissey C, et al.: Endocrine disrupting-chemicals and biochemical recurrence of prostate cancer after prostatectomy: A cohort study in Guadeloupe (French West Indies). Int J Cancer. 2020; 146(3): 657–663. PubMed Abstract \| Publisher Full Text\n\nKoutros S, Langseth H, Grimsrud TK, et al.: Prediagnostic Serum Organochlorine Concentrations and Metastatic Prostate Cancer: A Nested Case-Control Study in the Norwegian Janus Serum Bank Cohort. Environ Health Perspect. 2015; 123(9): 867–72. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChang ET, Boffetta P, Adami HO, et al.: A critical review of the epidemiology of Agent Orange/TCDD and prostate cancer. Eur J Epidemiol. 2014; 29(10): 667–723. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSorg O, Zennegg M, Schmid P, et al.: 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) poisoning in Victor Yushchenko: identification and measurement of TCDD metabolites. Lancet. 2009; 374(9696): 1179–1185. PubMed Abstract \| Publisher Full Text\n\nAbdallah MAE, Zaky AH, Covaci A: Levels and profiles of organohalogenated contaminants in human blood from Egypt. Chemosphere. 2017; 176: 266–272. PubMed Abstract \| Publisher Full Text\n\nVan den Berg M, Birnbaum L, Bosveld AT, et al.: Toxic Equivalency Factors (TEFs) for PCBs, PCDDs, PCDFs for Humans and Wildlife. Environ Health Perspect. 1998; 106(12): 775–792. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEroschenko VP: Estrogenic activity of the insecticide chlordecone in the reproductive tract of birds and mammals. J Toxicol Environ Health. 1981; 8(5–6): 731–742. PubMed Abstract \| Publisher Full Text\n\nMostafalou S, Abdollahi M: Pesticides and human chronic diseases: evidences, mechanisms, and perspectives. Toxicol Appl Pharmacol. 2013; 268(2): 157–77. PubMed Abstract \| Publisher Full Text\n\nQuagliariello V, Rossetti S, Cavaliere C, et al.: Metabolic syndrome, endocrine disruptors and prostate cancer associations: biochemical and pathophysiological evidences. Oncotarget. 2017; 8(18): 30606–30616. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHarris CA, Henttu P, Parker MG, et al.: The estrogenic activity of phthalate esters in vitro. Environ Health Perspect. 1997; 105(8): 802–11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee HR, Hwang KA, Choi KC: The estrogen receptor signaling pathway activated by phthalates is linked with transforming growth factor-β in the progression of LNCaP prostate cancer models. Int J Oncol. 2014; 45(2): 595–602. PubMed Abstract \| Publisher Full Text\n\nGajjar K, Marin-Hirsch PL, Martin FL: CYP1B1 and hormone-induced cancer. Cancer Lett. 2012; 324(1): 13–30. PubMed Abstract \| Publisher Full Text\n\nGo RE, Hwang KA, Choi KC: Cytochrome P450 1 family and cancers. J Steroid Biochem Mol Biol. 2015; 147: 24–30. PubMed Abstract \| Publisher Full Text\n\nGu CY, Qin XJ, Qu YY, et al.: Genetic variants of the CYP1B1 gene as predictors of biochemical recurrence after radical prostatectomy in localized prostate cancer patients. Medicine (Baltimore). 2016; 95(27): e4066. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrureau L, Multigner L, Hamdy F, et al.: PRISMA 2009 checklist.doc. figshare. Journal contribution. 2021. http://www.doi.org/10.6084/m9.figshare.14245694.v1" }	[ { "id": "125204", "date": "28 Feb 2022", "name": "Matthew J Roberts", "expertise": [ "Reviewer Expertise Prostate cancer - clinical research" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for asking me to review this systematic review. The topic is interesting, especially as many men are exposed to OCPs that may be implicated in PC pathogenesis. The literature is limited, which is acknowledged. For the most part, the authors have done a good job at including and discussing the relevant data from included studies.\nSome comments below that I hope improve the manuscript:\nGiven the paucity of data, no firm conclusions can be drawn. The authors have written to this for the most part, but some passages may overstate a potential association: - Abstract - \"However, the available evidence to date suggests the presence of a potential causal relationship between exposure to OPCs and PCa development and progression\" - this is unbalanced given the other half of the data showed no association. Could the authors please check the other potential passages and amend to reflect appropriately?\n\nThe results table is very brief, suggest expanding the results more than significant association so that readers can assess for themselves (e.g. the HRs seem much higher in the smaller studies, thus I would be more inclined to be influenced by the larger studies which are split in their conclusions)\n\nFigure 2 is ok but could be more informative - I would place a pathological/biological focus e.g. cancer spread and proliferation in between BCR (a clinical endpoint, I would also include metastasis); \"hormonal disorders\" is extremely vague, which of these are relevant to PC? it may also be helpful to show what downward effects these aspects cause to result in biological change\n\nThere is no limitations paragraph, there are many and so this should be included and lengthy. A major limitation is the assessment of association only, association does not equal causation in the absence of robust biological data. It does not appear from the paper content that such biological data exists. There are many causes for the associations seen, e.g. healthcare seeking behaviour, use of other medications (statins etc.). Also, men who are exposed may be more anxious, so more likely to opt for adjuvant radiotherapy after RP (before recent RADICALS/RAVES data..)\n\nI don't think that I could replicate this study as the exclusion reasons were not included. Could this be included as a supplementary table?\n\nSome minor spelling errors (e.g. \"Extracted domains included sttudy design.. and in Table 1 International Joural of Cancer..)\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Partly\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [ { "c_id": "7899", "date": "01 Mar 2022", "name": "Laurent BRUREAU", "role": "Author Response", "response": "Thank you for reviewing this work. The objective of this review is to warn against the risk of exposure to pesticides and in particular organochlorines on prostate cancer. Indeed, most of the data concern the impact of pesticides on the occurrence of cancer. However, there are few data on the impact of pesticides on the course and progression of the disease after diagnosis. This question is important because it is legitimate to think that prolonged exposure can have an impact on the progression of the disease. Our review, presents little data but it is the reality on the subject. We hope that this review will encourage other authors to carry out work on the same theme." } ] }, { "id": "172373", "date": "22 May 2023", "name": "Pradeep Kumar Sharma", "expertise": [ "Reviewer Expertise Environmental Carcinogenesis and hormone-dependent cancers" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nBrureau et al, in their work titled \"Organochlorine pesticide exposure and risk of prostate cancer development and progression: a systematic review\" have attempted to provide an evidence for causality between OCPs exposure and prostate cancer development, which is really pertinent in the current scenario where humans are being exposed to several different types of environmental chemicals that may affect their health in multiple ways including risk of hormone-dependent cancers such as breast and prostate cancer. While the authors have executed their work plan very nicely and covered all the articles available at the time of compilation of this study with appropriate exclusion/Inclusion criteria, yet I feel that the data insufficiency is a limitation in this study to derive concluding remarks on potential causal role of OCPs and PCa development. There are some comments that authors may consider for the comprehensiveness and better understanding the causal role of OCPs in PCa development.\nThe study compiles the data till July 2020, so authors should consider recent studies published in the subject area (e.g. PMID: 36949525 1, PMID: 36526827 2).\n\nWhile referring to the epidemiological studies in Table 1, authors should also indicate the exposure levels and/exposure window of OCPs (probable route of exposure to these OCPs in the cohorts).\n\nAuthors have restricted their study to a chemical group of OCPs only, while, given the fact that a number of other endocrine disrupting chemicals are routinely being identified, and known to have exposures in humans, it would be better to consider other potential EDCs for their causal relationship with PCa (preferably the epidemiological studies). It would give a comprehensive analysis of identifying substantial risk factors by considering the chemicals that are in frequent use as well, besides the OCPs.\n\nIn the introduction section, and accumulated environmental exposure to several hormones, it is not clear to what exogenous hormones human beings are exposed to? As exogenous substances (natural or synthetic) can mimic the action of natural ligands but are not the ligands for nuclear receptors particularly for estrogen- and androgen-receptors,\n\nCheck the clarity in this sentence in the introduction section \"However, to date there is a relative lack of information about the impact of exposure to OCPs upon on the development of aggressive metastatic PCa\",\n\nThough authors have mentioned several mechanistic reasons for these chemicals to cause PCa, their effects on androgen receptor (that is predominantly involved in the development of hormone-sensitive pCa) are largely missing and therefore authors should also consider this limitation in this study to highlight the limited understanding of OCPs like EDCs with respect to their androgen-mimicking potential.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-262
https://f1000research.com/articles/10-259/v1	30 Mar 21	{ "type": "Research Article", "title": "Attitudes of healthy volunteers to genetic testing in phase 1 clinical trials", "authors": [ "Sebastian Levesque", "Thomas M. Polasek", "Eric Haan", "Sepehr Shakib", "Sebastian Levesque", "Eric Haan", "Sepehr Shakib" ], "abstract": "Background: Genetic testing in clinical trials introduces several ethical and logistical issues to discuss with potential participants when taking informed consent. The aim of this study was to explore the attitudes of healthy volunteers in phase 1 studies to the topics of genetic security, genetic privacy and incidental genetic findings. Methods: Healthy volunteers presenting for screening appointments at a phase 1 clinical trial unit (CMAX Clinical Research, Adelaide, Australia) took an anonymous paper survey about genetic testing. Results: There were 275 respondents to the survey. The mean age was 27 years (range 18-73); 54% were male and 53% were of North/Western European ethnicity. Just over half the healthy volunteers thought genetic security (56%) and genetic privacy (57%) were “important” or “very important”. However, the security of their genetic information was ranked less important than other personal information, including mobile phone number, internet browser search history and email address. Two-thirds of respondents would trade genetic privacy for re-identifiability if information relevant to their health were discovered by genetic testing. Healthy volunteers favoured the return of incidental genetic findings (90% indicated this was “important” or “very important”). A level of risk (10 to 90%) for developing a serious medical condition that would “trigger” the return of incidental genetic findings to participants was not identified. Conclusions: Healthy volunteers screening for phase 1 clinical trials have mixed views about the importance of genetic security and genetic privacy, but they strongly favour the return of incidental genetic findings that could affect their health. These issues should be discussed with potential participants during informed consent for phase 1 clinical trials with genetic testing.", "keywords": [ "phase 1", "genetics", "healthy volunteers", "informed consent", "pharmacogenetics" ], "content": "Introduction\n\nGenetic testing during clinical drug trials can determine whether genetics influences how patients respond to drug therapy.1,2 Increasingly, genetic information is collected from healthy volunteers during phase 1 clinical studies to identify factors that inform later stages of drug development, including dosing strategies, patient selection, and the potential role of biomarkers to monitor drug responses.1\n\nGenetic testing in clinical trials introduces several ethical and logistical issues for participation, including concerns about genetic security and genetic privacy and how investigators should proceed with incidental genetic findings. Genetic security is the secure storage of genetic data and material. Clinical trial facilities and pharmaceutical companies must store these data for at least 15 years,3 so there is potential for misappropriation, release and misuse during this time. Genetic privacy is the protection of a person’s genetic information so they cannot be identified without knowledge and consent.4 Incidental genetic findings are results unrelated to the initial reason for genetic testing. These results may have implications for the current and future health and wellbeing of participants e.g., if genetic results influence susceptibility to disease or raise questions about ancestry or parentage. In Australia, the National Health and Medical Research Council (NHMRC) has recommendations on how to address genetic topics in clinical drug trials.5 For example, a separate participant information sheet and consent form (PICF) for the genetic component of studies should be used to discuss the nature of the genetic testing and its consequences for participants.\n\nThe attitudes of patients and the public to genetic testing in clinical trials have been explored in a very limited number of previous studies. These two groups generally favour the return of incidental genetic findings,6,7 but sometimes express concerns regarding genetic privacy and security, fearing disclosure of genetic information and widespread data sharing and misuse.8,9 There are no comparable data on these genetic topics for healthy volunteers. Given that healthy volunteers and patients are motivated differently for clinical trial participation,10,11 for example, healthy volunteers primarily focus on financial remuneration whilst patients do not, differences in attitudes towards genetic testing between the groups may be expected. Thus, the aim of this study was to explore the attitudes of healthy volunteers in phase 1 studies to the issues of genetic security, genetic privacy and the return of incidental genetic findings.\n\n\nMethods\n\nPotential participants being screened for studies at a clinical trial facility (CMAX Clinical Research Pty Ltd, Adelaide, Australia) were questioned about their attitudes to genetic testing using a paper survey (May to September 2019). No formal sample size was set, with as many potential participants approached during the study period as possible. The survey was de-identified and no personal information was collected apart from demographics. No potential biases were considered to influence participation and therefore the inclusion criteria were kept simple: participants were eligible if they were presenting for a healthy volunteer clinical drug study, were ≥18 years of age, and had sufficient English to allow informed consent and to answer the survey. There were no further inclusion or exclusion criteria. Informed consent was obtained by the lead author (S.L.) and participants completed the survey independently in the waiting room. The Human Research Ethics Committee at the University of Adelaide approved the study (H-2019-085).\n\nSurveys were written by the authors to investigate attitudes towards genetic security, genetic privacy and incidental genetic findings. Questions were written in non-technical language to maximise readability and understanding. No formal validation or piloting of the survey was conducted prior to enrolment of the first participant. There were two iterations of the survey: an initial version (1st survey) involving Q1-6 and then an updated version (2nd survey) that included three additional questions (Q7-9) (see extended data12). Several question formats were used. A 5-point Likert scale included the options “very unimportant”, “unimportant”, “neutral”, “important” or “very important”. Other questions used a rank scale method (1-5, with 1 being the least important and 5 being the most important), and in the case of ties, a mean rank was ascribed to the responses. Polar responses (yes/no) were used for statements regarding incidental genetic findings. The final question format was a visual analogue scale to investigate attitudes towards genetic privacy. The demographic data collected were age, ethnicity (2nd version of the survey only), level of education, and the number of clinical trials undertaken previously at CMAX Clinical Research. The definitions of ethnicity were those used by the Australian Standard of Classification of Ethnic and Cultural Groups.13\n\nData from paper surveys were transcribed electronically and analysed using SPSS v25 (IBM corporation, Armonk, NY). Some responses were dichotomised as “favoured” (comprising the responses “important” and “very important”) or “not favoured” (comprising the responses “very unimportant”, “unimportant” and “neutral”). After analysis of the first 190 surveys, an amended survey was created which included three new questions. These questions created scenarios to discern the motivations for some responses. A sample size of 300 was calculated to result in a 95% confidence interval of 5-10% for a range of proportions between 30%-70% for each question. Potential statistically significant relationships between responses and demographics were examined. Kruskal-Wallis tests were performed for any relationships with age, number of previous CMAX studies, ethnicity, and education. Mann-Whitney U-tests were performed for any relationships with gender. Statistical significance was set at p < 0.05.\n\n\nResults\n\nTable 1 shows the demographic results of the study. There were 275 respondents who completed both surveys (85.8% participation) – 189 for the first and 85 for the second iteration. The mean age was 27 years (range 18-73 years); 54% were male, 55% completed high school as their highest form of education, 62% had not undertaken a clinical trial at CMAX Clinical Research previously, and 53% were of North/Western European ethnicity (Table 1).16,17\n\nJust over half the participants had a favourable attitude towards the importance of genetic security (56%) (Table 2 and Figure 1A). The favourable response was significantly associated with younger age (p=0.017) and the median age of favoured responses was 26 years. When participants were asked to rank the security of their genetic information against other personal identifying information, genetics had a similar mean rank to medical history, and was ranked less important than the other personal information such as mobile phone number, internet browser search history and email address (Figure 1B). There were no significant associations between this ranking question and demographics (Table 2).\n\nMore than half the participants had a favourable attitude towards the importance of genetic privacy (57%) (Table 2 and Figure 2A). No demographic factors were associated with responses to this question. When choosing between genetic privacy and re-identifiability, most respondents (67%) chose an option in the re-identifiability portion of the visual analogue scale (Figure 2B). Women were more likely to prefer re-identifiability than men (median on response scale of 0.70 versus 0.47, p=0.033), but no other statistical relationships to demographics were found (Table 3).\n\nParticipants strongly preferred the return of incidental genetic findings (90% favoured) and wanted this information included in PICFs (87% favoured) (Table 2) (Figure 3A). Both questions were not associated with any statistically significant relationships to demographics. Respondents also preferred the return of incidental genetic findings for several cancer scenarios with varying hypothetical risks and treatability, but they were slightly less interested if the risk was very low and the cancer was untreatable (Figure 3B). When participants were asked to choose a risk for a specific disease that should trigger the return of incidental genetic findings, answers were similarly divided across all levels of risk (10-90%). The three most popular answers were for the options of 90% risk for developing a disease, with cancer > heart disease > neurodegeneration (Figure 3C). Older participants were more likely to select higher risk of disease to trigger the return of incidental genetic findings, whereas respondents with more clinical trial experience studies wanted information provided at lower levels of risks (Table 3). About 90% of participants wanted information provided to them in the future if sample re-analysis or new genetic discoveries could influence their health and wellbeing (Table 2).\n\n\nDiscussion\n\nThis is the first study to report the attitudes of healthy volunteers to genetic testing in phase 1 clinical trials. These attitudes are important to understand given the rise of genetic testing in early drug development and the unique ethical and practical aspects of testing healthy volunteers who typically have few interactions with healthcare providers. Here, the focus was the genetic testing issues previously explored in limited studies with patients and the public – genetic security, genetic privacy, and the return of incidental genetic findings.6–11\n\nThere was relative indifference towards genetic security, with healthy volunteers ranking the importance of this topic lower than for other personal information. Indeed, mobile phone number and email address are provided to the clinical trial site during screening, so for genetic security to rank lower than these two pieces of information emphasises the relative indifference to the topic. This is similar to limited data in patients, with only 35% of potential biobank participants and just one patient in a focus group of 15 with epilepsy expressing concerns about genetic security.8,14\n\nRegarding genetic privacy, healthy volunteers also held mixed opinions, with just over half considering it “important” or “very important”. This is comparable to a survey of 4659 US adults from the public, in which 44% would protect genetic test results, whereas the others were happy to share their genetic data with researchers.15 Additionally, a survey assessing the views of 57 patients to pharmacogenomic testing showed that approximately 40% expressed concerns about privacy.9 When asked whether they would trade privacy for re-identifiability in the future, respondents to our survey overwhelmingly favoured re-identifiability of their genetic information. Taken together, these findings support clinical research governance that de-identifies genetic information to maintain privacy, but with mechanisms in place to re-identify that information if asked by clinical trial participants.\n\nThe healthy volunteers wanted incidental genetic findings returned to them, and this applied to a range of hypothetical clinical scenarios with varying degrees of disease risk. Older participants and those with more clinical trial experience accepted higher disease risk before triggering the return of such information. Exactly why these two groups indicated higher disease risk is unclear, but it may be simply related to life experience and greater acceptance of the unknown. The majority of oncology patients in a previous study also favoured the return of incidental findings, even if they constituted “bad news”, such as high risk of developing an untreatable cancer.6 Studies of the public indicate the same (4961 participants), with “treatability” being an important consideration and with 98% in favour of findings being returned for “a serious disease that is life threatening but could be prevented”.7 Together with the results in healthy volunteers, these data suggest that everyone should be asked about their wishes for the return of incidental genetic findings when being screened for clinical trials.5\n\nThere are several limitations of the study. First, it was conducted at one site, so care is required in extrapolating the results to other phase I clinical units with significantly different volunteer demographics. Second, the survey was offered opportunistically to all people in the waiting room, independent of the type of clinical trial screening. It is possible that attitudes to genetic testing are different between healthy volunteers being screened for clinical trials with genetic testing and those being screened for clinical trials without genetic testing. Third, the study did not assess baseline “genetic literacy”, so misunderstanding of questions is possible, particularly the conceptually more difficult questions related to disease risks. Lastly, the study was not designed to capture the nuances of genetic testing because the topic is too broad and complex to be condensed into a short survey e.g., the specifics of testing.\n\nIn conclusion, healthy volunteers who are screening for phase 1 clinical trials have mixed views about the importance of genetic security and genetic privacy, but they strongly favour the return of incidental genetic findings that could affect their health. These issues should be discussed with potential participants during informed consent for phase 1 clinical trials with genetic testing.\n\n\nData availability\n\nFigshare: Attitudes to Genetic Testing in Phase 1_Survey Data. https://doi.org/10.6084/m9.figshare.14204507.v1.16\n\nFigshare: Untitled Attitudes to Genetic Testing in Phase 1_Amalgamated Survey Data. https://doi.org/10.6084/m9.figshare.14204516.v1.17\n\nFigshare: Attitudes to genetic testing in phase 1 survey questions. https://doi.org/10.6084/m9.figshare.14182898.v1.12\n\nThis project contains the following extended data:\n\n- A supplementary figure showing all the survey questions used in the study.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nWe thank the staff at CMAX Clinical Research Ltd Pty for assisting with this study.\n\n\nReferences\n\nBurt T, Dhillon S: Pharmacogenomics in early-phase clinical development. 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PubMed Abstract \| Publisher Full Text\n\nNational Health and Medical Research Council: Principles for the translation of ‘omics’– based tests from discovery to health care.2015.\n\nGray SW, Park ER, Najita J, et al.: Oncologists’ and cancer patients’ views on whole-exome sequencing and incidental findings: results from the CanSeq study. Genet Med. 2016; 18(10): 1011–1019. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMiddleton A, Morley KI, Bragin E, et al.: Attitudes of nearly 7000 health professionals, genomic researchers and publics toward the return of incidental results from sequencing research. Eur J Hum Genet. 2016; 24(1): 21–29. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRahm AK, Wrenn M, Carroll NM, et al.: Biobanking for research: a survey of patient population attitudes and understanding. J Community Genet. 2013; 4(4): 445–450. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLemke AA, Hulick PJ, Wake DT, et al.: Patient perspectives following pharmacogenomics results disclosure in an integrated health system. Pharmacogenomics J. 2018; 19(4): 321–331. PubMed Abstract \| Publisher Full Text\n\nFacio FM, Brooks S, Loewenstein J, et al.: Motivators for participation in a whole-genome sequencing study: implications for translational genomics research. Eur J Hum Genet. 2011; 19(12): 1213–1217. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTreloar SA, Morley KI, Taylor SD, et al.: Why do they do it? A pilot study towards understanding participant motivation and experience in a large genetic epidemiological study of endometriosis. Community Genet. 2007; 10(2): 61–71. PubMed Abstract \| Publisher Full Text\n\nPolasek TM: Attitudes to genetic testing in phase 1 survey questions. figshare. Figure. 2021. Publisher Full Text\n\nAustralian Bureau of Statistics: Australian Standard Classification of Cultural and Ethnic Groups.2016.\n\nMcGuire AL, Hamilton JA, Lunstroth R, et al.: DNA data sharing: research participants' perspectives. Genet Med. 2008; 10(1): 46–53. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKaufman DJ, Murphy-Bollinger J, Scott J, et al.: Public opinion about the importance of privacy in biobank research. Am J Hum Genet. 2009; 85(5): 643–654. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPolasek TM: Attitudes to Genetic Testing in Phase 1_Survey Data. figshare. Dataset. 2021. Publisher Full Text\n\nPolasek TM: Attitudes to Genetic Testing in Phase 1_Amalgamated Survey Data. figshare. Dataset. 2021. Publisher Full Text" }	[ { "id": "82525", "date": "06 Apr 2021", "name": "Martin D. Lewis", "expertise": [ "Reviewer Expertise Genetics", "neuroscience", "cell biology", "major depressive disorder and pharmacogenomics." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors address important aspects of genetic testing, namely participant concerns regarding data security, privacy and incidental findings. As most participants were found to favour being informed of incidental findings, this should influence future consent options for study participants. The number of studies involving genetic testing is increasing, with this fundamental component of the ethics process critically important.\nThe article is well written, with the study, survey design, and analysis very clearly and appropriately described. The literature cites the current national guidelines and standards, in addition to relevant publications in the manuscript focusing on questions addressed in this field. All data was made available ensuring reproducibility. The conclusions draw from the data are adequately supported.\nMinor changes: Page 4 - The sentence under Demographic stating, “There were 275 respondents that completed both surveys…” could be misunderstood as some respondents completed two surveys. This could be altered to “There was a total of 275 respondents that completed the first (189) or second (85) iteration of the survey”.\n\nThere appears to be an inconsistency with the previous paragraph under ‘Data Analysis’ that says after analysis of the first 190 surveys, a second iteration was created. Were 190 survey of the first iteration started and only 189 completed?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "119428", "date": "24 Jan 2022", "name": "Colleen Aldous", "expertise": [ "Reviewer Expertise Healthcare research with a special interest in rare and congenital disorders." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nHealthy participants were selected to complete an unpiloted and non-validated questionnaire which investigated attitudes to genetic information security, privacy and the return of incidental genetic findings. Ethics permission was provided and informed consent was noted. This is a descriptive study and the data is presented appropriately. The Lickert scale data is converted into a form which can be statistically compared. The findings are clearly summarised. There is a conclusion based on the data that the return of incidental genetic findings should be addressed. The limitations of the study are stated.\nQueries:\nA sample size of 300 participants had been calculated, however the final number reported on was 275. Could the authors please clarify this point?\n\nEthnicity is addressed but seems not to have much of a role in the answering of the research questions. In the abstract the reader is left questioning the other ethnicities after only North/Western European ethnicity is mentioned. In Table 1 the ethnicity of only 84 participants appears to have been reported. I would suggest that the authors state that ethnicity, for this study, was not addressed.\n\nThis study raises further questions for future studies.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-259
https://f1000research.com/articles/9-1250/v1	16 Oct 20	{ "type": "Case Report", "title": "Case Report: Thalamomesencephalic stroke due to vasculitis in a patient with HIV", "authors": [ "Jerry George", "Sibi Joseph", "Mongezi Tau", "Lourdes de Fatima Ibanez Valdes", "Thozama Dubula", "Humberto Foyaca-Sibat", "Jerry George", "Sibi Joseph", "Mongezi Tau", "Lourdes de Fatima Ibanez Valdes", "Thozama Dubula" ], "abstract": "We present a 41-year-old HIV-positive female patient complaining of complete right palpebral ptosis, diplopia, and inability to balance herself. On examination, the right eye was able to move laterally and downwards. The motor exam showed left hemiparesis (4/5) on upper and lower limbs, bilateral Babinski sign with left hemiataxia without the sensory disorder. A computed tomography (CT) angiogram confirmed a diffuse vasculitis with parenchymal changes in the right thalamus and midbrain. CT scan and magnetic resonance imaging angiography demonstrated an ischemic infarct on the right paramedian branch of the posterior cerebral artery territory. This patient did not present clinical manifestations of the thalamic lesion. To our knowledge, this is the first reported case of a young patient presenting a unilateral thalamomesencephalic ischemic stroke secondary to HIV vasculitis with bilateral Babinski signs and without thalamic signs in the medical literature.", "keywords": [ "Thalamomesecephalic stroke", "HIV", "Vasculitis" ], "content": "Introduction\n\nIn 2018, Sato et al. reported a 62-year-old man presenting with a rare eye movement. This patient had a vertical one-and-a-half syndrome caused by unilateral thalamomesencephalic stroke (TMS)1. Other eye movements' abnormalities, such as bilateral vertical gaze palsy, were previously reported due to a unilateral stroke of the rostral midbrain by other authors2,3.\n\nThe anatomical circulation of the brain is complex and diverse, and the circle of Willis variations can include absence or fusion of components, incomplete process, fenestrations, fetal branches, and asymmetrical and duplication. From previous studies on autopsy and structural imaging scans, normal anatomical variations were detected in 48–58% of the general population, and even during fetal development4–8.\n\nAn investigation done on human cadaveric brains have been demonstrated four major thalamic arterial territories, with notable blood variations. These areas receive blood supply by the polar, paramedian/thalamoperforating arteries, thalamogeniculate, and posterior choroidal arteries9–17. The perforating arteries supply the medial the walls of the third ventricle, hypothalamus, and subthalamic-mesencephalic junctions. These areas include the oculomotor nucleus, red nucleus, subthalamic nucleus, substantia nigra, pretectum, trochlear nucleus, reticular formation of the midbrain, posterior part of the internal capsule, the rhomboid fossa, and also the rear part of the thalamus9–20. Because the artery of Percheron occlusion can affect the thalamus and midbrain at the same time, here we have to mention that artery of Percheron is an uncommon vascular variant of the paramedian branches of the posterior cerebral artery, arising from one P1 segment, bifurcates, and bilateral supply to the bilateral paramedian thalami and the rostral midbrain10,11 but not unilaterally. Therefore, occlusion of Percheron arteriole causes an atypical pattern of bilateral infarct of the median thalami with or without mesencephalic damage.\n\nFrom 2010 and 2017, several authors21–26 also reported a case series of ischemic stroke on the thalamus and different clinical manifestations. Other clinical presentations of TMS include see-saw nystagmus that shows intorsion and elevation of one eye, with synchronous extorsion and depression on the contralateral one, convergence-retraction nystagmus and contraversive ocular tilt reaction probable due to ischemic involvement of the interstitial nucleus of Cajal27, anisocoria. Another author found vertical ocular motor disturbances in the vertical plane and eye movement synkinesis, hypersomnia, and coma as a clinical manifestation of TMS28. Others reported headaches, blurred vision, and diplopia as a particular variant of cerebral lacunae TMS29. In 2012, Benjamin et al. established that HIV infection can cause TMS by opportunistic infections, secondary to a cardioembolic phenomenon, coagulopathy, and vascular diseases such as stenosis, acquired aneurysm, vasculitis, and direct/indirect effect of HIV infection and antiretroviral therapy30. In our region, ischemic stroke due to infectious vasculitis is quite common. In 2017, the first case presenting bi-thalamic infarctions leading to acute vascular dementia associated with HIV infection was reported31.\n\n\nCase presentation\n\nA 41-year-old female presented with a 5-day history of inability to open the right eye associated with decreased vision of the right eye, which subsequently developed binocular diplopia. The patient also reported a failure to balance herself and could not walk independently. There was no history of trauma, excessive use of NSAIDs, contraceptives, use of vitamin supplements, or complaint of headaches. The patient did not smoke, drink alcohol, or use other recreation or illicit drugs. The patient has a background history of hypertension since her last pregnancy in 2016 and has been on treatment with hydrochlorothiazide (12.5 mg daily) and enalapril (5 mg daily). HIV-reactive with the latest CD4 (01/2020) count of 715 and viral load are lower than the detectable limit on treatment with a combination of tenofovir/emtricitabine/efavirenz TDF/FTC/EFV (300/200/600 mg daily).\n\nOn the nervous system examination, the patient was alert and well oriented with no meningeal signs. A cranial nerve exam revealed right cranial nerve 3rd palsy, right complete ptosis, right mydriatic pupil nonresponsive to light, and paralysis of the medial, superior, and inferior rectus plus inferior oblique (Figure 1).\n\nThe motor exam showed left hemiparesis (4/5) on upper and lower limbs, bilateral Babinski sign with left hemiataxia despite muscle weakness on the affected side, and no sensory disorder or extrapyramidal signs. The rest of the examination was within normal limits.\n\nThe investigations done were as follows: Blood tests (on the day of admission) See Table 1\n\nComputed tomography (CT) angiogram and MRI (done two days after admission) showed diffuse vasculitis with parenchymal changes seen in the right thalamus and midbrain and ischemic infarct in the area supplied by the right paramedian branch of the posterior cerebral artery due to vasculitis (Figure 2 and Figure 3).\n\nThe axial view shows the right hyperdense lesion at the paramedian thalami caused by ischemic infarct secondary to HIV vasculitis.\n\nThe axial view shows a hyperdense lesion on the right midbrain caused by ischemic stroke due to HIV vasculitis.\n\nThe cardiology team requested a cardiac review. A cardiac ultrasound (done the day after admission) showed an ejection fraction of 75%, No valvulopathy or effusion was present. The patient was admitted and started on the following treatment: Vitamin B12 supplementation (1000 µg IM daily for five days in the first week, then weekly for five weeks, aspirin (150 mg daily), enoxaparin (40 mg s/c daily), simvastatin (20 mg daily), pyridoxine (50 mg daily), thiamine (100 mg daily). The patient continued the chronic medication (hydrochlorothiazide 12.5 mg, enalapril 5mg and TDF/FTC/EFV 300/200/600 mg daily). Physiotherapy and occupational therapy are actively working with the patient.[TJ4] The patient received rehabilitation in our ward for two weeks. The right-sided hemiataxia did improve, but the power on the right side was still 4/5. She was referred to her base hospital to continue rehabilitation and a follow-up date with us in 1 month.\n\n\nDiscussion\n\nHere we report a case of a 41-year-old woman with right-sided TMS. Unilateral TMS is uncommon, and its incidence remains unknown, but one study showed that it comprises about 0.6% to 1% of midbrain ischaemic strokes and often accompanied by other posterior circulation infarcts32.\n\nBaran et al. conducted an observational study in 2018, which showed a male predominance. The study also showed that from an etiological point of view, the most common cause was extensive atherosclerosis, followed by cardio-embolism, apart from small vessel disease33.\n\nThe main risk factors associated with extensive atherosclerosis are hypertension, diabetes, hyperlipidemia, smoking, and previous history of stroke. The main risk factors for cardio-embolism in these patients is atrial fibrillation33. Patients who are suffering from peripheral vascular disease and coronary artery disease are at risk34.\n\nHIV is a risk factor for stroke35 and is associated with advanced disease36. There have been numerous mechanisms proposed to explain this. A systematic review done by Addallah et al.37 reported that this could be due to HIV-associated opportunistic infections, HIV-induced coagulopathy, and chronic inflammatory processes that can accelerate atherosclerosis. Another systematic review by Bogorodskaya et al.38 also reported that some antiretrovirals (lopinavir, indinavir, and abacavir) were also associated with an increased risk of stroke.\n\nLesions of the midbrain can present as distinct syndromes. However, because of the structures' close organization, there can be considerable overlap of these syndromes. The neurological manifestation will depend on which area of the midbrain is affected and whether one half or both halves are involved and whether adjacent structures (thalamus, pons, cerebellum) are also involved. The symptoms may include but are not limited to, low equilibrium, weakness of one or both sides of the body, diplopia, and slurred speech39. The most common examination findings include ataxia, limb weakness, dysarthria, sensory disturbance, oculomotor findings (3rd nerve palsy, internuclear ophthalmoplegia), and dysarthria40. The exact pattern will depend on the area involved and whether surrounding structures are also involved (thalamus, pons, medulla, etc.). There are have been midbrain syndromes, which include, among others, Weber syndrome, Claude's syndrome, Nothnagel syndrome, and Benedikt's syndrome. Benedikt's syndrome presents with a contralateral rubral tremor, which she does not have.\n\nWeber syndrome is a result of a lesion involving the ventromedial area of the midbrain. They present with ipsilateral 3rd nerve palsy with contralateral hemiplegia. Our patient has Claude's syndrome, which presents ipsilateral 3rd nerve palsy with contralateral cerebellar ataxia due to the dorsal tegmentum lesion, which involves the 3rd nerve nucleus/fibers and also involving either the red nucleus, superior cerebellar peduncle, or brachium conjunctivum37. Benedikt's syndrome is due to a lesion involving the tegmentum. It presents with ipsilateral 3rd nerve palsy and contralateral ataxia, but there is also the involvement of the fibers of the corticospinal tract and will result in contralateral hemiparesis even41. When assessing these kinds of patients, it is essential to ascertain a good history and physical examination and check the National Institute for Health Stroke Score42. Imaging to confirm the diagnosis is mandatory. CT or MRI angiogram is usually requested to identify the stenosed vessels or identify other possible vascular problems. Blood workup for stroke is compulsory, which includes but is not limited to full blood count, renal function tests, international normalized ratio, lipid profile, HIV ELISA, and if young to include thrombophilia screen, Antinuclear antibodies, and glycosylated hemoglobin. ECG to rule out possible atrial fibrillation and transthoracic or even transesophageal echocardiography to identify cardiac causes.\n\nThe management approach depends on the etiology of the stroke. If the infarct is ischaemic, the reviewed literature recommends thrombolysis if posterior circulation strokes meet the established criteria.43. Mechanical thrombectomy benefits are not yet well established, but it can be done44. Then after the acute period, it is crucial to managing the risk factors and causes. Then treat the risk factors such as arterial hypertension, diabetes mellitus, hyperlipidemia, and secondary prophylaxis. If there is a cardiac cause, then it should be treated. A multidisciplinary approach is vital for patients presenting TMS. The stroke team should include dieticians, physiotherapy, speech therapy, occupational therapy, and social workers apart from the medical specialists.\n\nRisk factors for developing stroke in our patient were hypertension, HIV, and hyperlipidemia. The patient also had contralateral hemiparesis and hemiataxia with bilateral Babinski and hyperreflexia on both lower limbs. Her blood workup showed that she was virally suppressed and had hyperlipidemia. The MRI and CT angiograms showed evidence of an infarct involving the ventromedial midbrain and thalamus, and in the absence of other lesions. Cardiovascular investigations ruled out a cardiac source of the infarct. In this patient, hypertension, HIV infection, and hyperlipidemia predisposed her to the stroke. The patient is markedly younger than one would typically expect for a TMS (median age around 64 years)32. Of note in our patient is the presence of a bilateral Babinski sign, which never happens in a patient with Claude's syndrome.\n\nGenerally, the strokes involving posterior circulation have a higher mortality rate than those involving anterior circulation unless it involves the smaller blood vessels41, as happened in our case. The present case is unique, among other reasons, owing to the bilateral Babinski sign and the absence of thalamic manifestations without other lesions affecting different segments of the brainstem and the spinal cord. The patient's age (41 years) also makes this case uncommon. The patient's leading risk factor is HIV vasculitis, which has not been implicated for TMS from the literature reviewed. We did not find signs of middle longitudinal fascicle (MLF) typical syndrome. MLF syndrome, secondary to ischemic stroke affecting only the mesencephalon, is a rare occurrence45,46. We would highlight that we could not find the cause of the bilateral Babinski signs in this case. This patient did not present thalamic characters despite the ischemic lesion in the right thalamus despite the midbrain's role over the thalamus. The modulation of thalamic neurons is necessary to control adaptive behavior mediated by midbrain cholinergic transmission. The central cholinergic neurons in the mesencephalon are at the pedunculopontine nucleus and the laterodorsal tegmental nucleus, which provide dense innervation of the thamalus. Recently, Huerta-Ocampo et al. confirmed that midbrain cholinergic neurons could innervate all thalamic nucleus. They also found that these axons topographically are well organized and provide a segregated innervation of the thalamic nuclei47. Therefore, we expected some evidence of thalamic dysfunction in this patient. However, these ischemic lesions on the midbrain did not cause abnormal behavior or thalamic manifestations in our patient, which is a novel finding.\n\nTo our knowledge, this is the first patient presenting with unilateral TMS secondary to HIV vasculitis with bilateral Babinski signs, and without thalamic manifestations to be reported in the medical literature. In young patients presenting with unilateral TMS, HIV vasculitis is one of the etiological diagnoses to be considered. Still, we recommend an extensive investigation based on a series of cases to support this postulate.\n\n\nData availability\n\nAll data underlying the results are available as part of the article, and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patient.", "appendix": "Author contributions\n\n\n\nAll authors contributed equally to the elaboration of this manuscript. MT and SJ collected data and planning this report, JG and LIV wrote the first draft and reviewed bibliographically, TB and HFS wrote the final manuscript. All authors reviewed the final manuscript, made corrections, and agreed for publications\n\n\nAcknowledgment\n\nWe wish to thank Dr. M Anwary from the Department of Radiology. Nelson Mandela Academic Central Hospital Mthatha, South Africa, for the investigations done.\n\n\nReferences\n\nSato K, Takahashi Y, Matsumoto N, et al.: Rare valiant vertical one-and-a-half syndrome without ipsilateral upward gaze palsy in a patient with thalamomesencephalic stroke. Neurol Clin Neurosci. 2018; 6(5): 133–135. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPierrot-Deseilligny CH, Chain F, Gray F, et al.: Parinaud's syndrome: electro-oculographic and anatomical analyses of six vascular cases with deductions about vertical gaze organization in the premotor structures. Brain. 1982; 105(Pt 4): 667–96. 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PubMed Abstract \| Publisher Full Text\n\nRuchalski K, Hathout GM: A medley of midbrain maladies: a brief review of midbrain anatomy and syndromology for radiologists. Radiol Res Pract. 2012; 2012: 258524. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKhimKwah L, Diong J: National Institutes of Health Stroke Scale (NIHSS). J Physiother. 2014; 60(1): 61. PubMed Abstract \| Publisher Full Text\n\nFilho JO, Samuels OB: Approach to reperfusion therapy for acute ischemic stroke.2020. Reference Source\n\nPowers WJ, Rabinstein AA, Ackerson T, et al.: Guidelines for the Early Management of patients with acute ischemic stroke: 2019 Update to 2018: A Guideline for Healthcare Professionals from the American Heart Association/American Stroke Association. Stroke. 2019; 50(12): e344–e418. PubMed Abstract \| Publisher Full Text\n\nYeo SS, Jang SH, Kwon JW, et al.: Three-Dimensional Identification of the Medial Longitudinal Fasciculus in the Human Brain: A Diffusion Tensor Imaging Study. J Clin Med. 2020; 9(5): 1340. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKochar PS, Kumar Y, Sharma P, et al.: Isolated medial longitudinal fasciculus syndrome: Review of imaging, anatomy, pathophysiology, and differential diagnosis. Neuroradiol J. 2018; 31(1): 95–99. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHuerta-Ocampo I, Hacioglu-Bay H, Dautan D, et al.: Distribution of Midbrain Cholinergic Axons in the Thalamus. eNeuro. 2020; 7(1): ENEURO.0454-19.2019. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "73256", "date": "30 Oct 2020", "name": "Jefferson V. Proano", "expertise": [ "Reviewer Expertise Neurology and Infection disease of the nervous system" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors assume that the etiology of the strokes in the case they present, is due to vasculitis due to HIV, but it is completely necessary to exclude other causes of vasculitis, such as systemic lupus erythematosus, in addition to the fact that the patient has other risk factors for strokes. Furthermore, it is difficult to suppose that this patient having the HIV virus copy number parameter at levels of undetectable, however, develops vasculitis due to this cause.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [ { "c_id": "6085", "date": "02 Nov 2020", "name": "Humberto Foyaca-Sibat", "role": "Author Response", "response": "Dear Reviewer, We confirmed the diagnosis of vasculitis by MRI images, as you can see in the radiologist's report. In this region, the commonest cause of vasculitis is an infectious disease caused by 1.- HIV/AIDS, 2.- TB, 3.- Neurocysticercosis, or 4.- Neurosyphilis. Fortunately, we did not see SLE causing vasculitis at this shores. Because we could not rule out SLE, then we did not say HIV-vasculitis, then we titled this manuscript as....vasculitis in HIV patient. Thanks for your kind attention and professional comments. Regards, Prof H Foyaca. MD Ph.D" }, { "c_id": "6257", "date": "11 Jan 2021", "name": "Humberto Foyaca-Sibat", "role": "Author Response", "response": "Dear Reviewer, As you can see we made all the changes that you suggested. We are completely agreed and very happy with your suggestions. Thanks a lot for your kind attention and professional support. Regards, Dr. Foyaca" } ] } ]	1	https://f1000research.com/articles/9-1250
https://f1000research.com/articles/10-139/v1	23 Feb 21	{ "type": "Research Article", "title": "MGMT promoter gene methylation and neurological scale improvement in glioma: a cohort study", "authors": [ "Pricilla Yani Gunawan", "Andi Asadul Islam", "Julius July", "Ilhamjaya Patelongi", "Agussalim Bukhari", "Muh. Nasrum Massi", "Andi Asadul Islam", "Julius July", "Ilhamjaya Patelongi", "Agussalim Bukhari", "Muh. Nasrum Massi" ], "abstract": "Background: Glioma is one of the most common primary brain tumours and conveys a dismal prognosis despite aggressive treatment. Several biomarkers have been studied in the hope of yielding better diagnostic accuracy and improving patient management. Besides survival, functional and neurological disability are concerns that have no lesser importance. In 2017, a disease-specific assessment tool – the Neurologic Assessment in Neuro-Oncology (NANO) scale – was developed to measure neurologic function in neuro-oncology cases. We sought to determine biomarkers that might be associated with neurological scale improvement in glioma patients. Methods: Glioma grade II-IV patients were recruited from three major hospitals in Jakarta-Tangerang. Isocitrate dehydrogenase (IDH) mutation and O6-methylguanine-DNA methyltransferase (MGMT) promoter gene methylation were tested, as well as patients’ neurological function before surgery and three months after. Improvement in neurological scale (NANO scale) was considered positive if there was a decrement of ≥1 of the scale. Results: There were 54 patients included in the study. Mean age was 43.63 (14.723) years old, and 61.1% were male. As much as 16 (29.6%) carried a mutation in codon 132 of the IDH1 gene, and 33 (61.1%) were MGMT methylated. Median NANO scale score before and three months after surgery was 4 (0-12) and 3 (0-12), respectively. Neurological improvement was found in 44 (81.5%) of the patients. Among patients with MGMT promoter gene methylation, 90.9% showed neurological improvement (p=0.035; OR=5; 95%CI 1.122-22.272). Conclusions: Gliomas with MGMT promoter gene methylation are more likely to show neurological improvement three months after surgery.", "keywords": [ "MGMT promoter gene methylation", "Glioma", "Neurological Scale", "NANO Scale" ], "content": "Introduction\n\nGlioma is one of the most common primary brain tumours, in which 80% of the cases are found to be malignant (Alifieris & Trafalis, 2015; Schwartzbaum et al., 2006; Stupp et al., 2010). Grade II glioma has a median survival ranging from four to more than ten years (Bell et al., 2018; Claus & Black, 2006). However, glioblastoma – a grade IV glioma – accounts for the majority of gliomas and carries a poor prognosis with median survival of six to 14 months despite aggressive treatment with radiotherapy plus chemotherapy, and less than 12 months with radiotherapy alone (Stupp et al., 2005).\n\nMolecular characteristics of glioma have undergone extensive research in the last decade, and some biomarkers have even been integrated into the 2016 glioma classification. Isocitrate dehydrogenase (IDH) mutation and O6-Methylguanine-DNA methyltransferase (MGMT) promoter gene methylation has been associated with better survival. Nonetheless, patients’ quality of life and neurological deficit have not been considerably measured and correlated with these prognostic biomarkers.\n\nIn 2017, the Neurological Assessment of Neuro-Oncology (NANO) scale was developed to objectively measure neurological deficit in patients with brain tumours (Nayak et al., 2017). Previous studies have shown that a good initial NANO scale score was significantly associated with improvement in neurological deficit two months after surgery (Gunawan et al., 2020) and a more powerful method to predict prognosis during initial diagnosis and disease progression (Lee et al., 2018). In this study, we aimed to elucidate biomarkers that might be associated with neurological scale improvement three months after surgery.\n\n\nMethods\n\nThis is a cohort study, with a purposive sampling method. There were 72 suspected brain tumour patients, aged more than ten years old, who presented at three major hospitals in Jakarta and Tangerang over a period of one year (July 2019–July 2020). After a brain MRI, 5 patients were excluded due to tumour location (brainstem or infratentorial). Written consent was obtained from every patient or their caregiver (for patients who are unable to give consent). Karnofsky Performance Scale (KPS) as well as neurological scale were assessed before surgery. Following tumour resection, 62 patients were histologically confirmed as having glioma grade II-IV. Tumour tissue was tested for IDH mutation as well as MGMT promoter gene methylation. Patients underwent standardised therapy, and neurological function was re-assessed three months after surgery. Six patients were deceased before the second neurological scale assessment, and two patients were lost to follow-up, hence were excluded from the study. The study was approved by Medical Ethical Research Committee, Universitas Hasanuddin, No: 1232/UN4.6.4.5.31/PP36/2019.\n\nHistopathological and molecular analysis was carried out in Mochtar Riady Institute Tangerang and Kalgen Innolab Clinical Laboratory, Jakarta. Tissue sample dissected from the tumor was immediately put in formaldehyde 10% buffer. Six to 24 hours later, the desired tumor tissue was stored into cassettes and processed in an automatic tissue processor. After an overnight processing, tissue was put into a mold with paraffin wax. Paraffin block with the embedded tissue was cut using a microtome and placed on glass slides stained with hematoxylin and eosin to determine the diagnosis and grade of malignancy.\n\nGenomic DNA was extracted from paraffin-embedded tumor tissues using the QIAamp®DNAMicroKit (QIAGEN, catalogue number 56304) following the manufacturers’ protocol.\n\nIDH mutation evaluation was done using high resolution melting (HRM) analysis and Sanger sequencing of polymerase chain reaction (PCR)-amplified fragments, which were generated during the HRM procedure with the PCR primers. Primers used were: IDH1-forward 5’-CGGTCTTCAGAGAAGCCATT-3’ and IDH1-reverse 5’-GCAAAATCACATTATTGCCAAC-3’ (gBlocks Gene Fragment, Integrated DNA Technologies).\n\nHRM analysis consists of 3 steps: PCR reaction, DNA melting, and data analysis. These analyses were performed using the LightCycler® 480 High-Resolution Melting Master Kit (Roche) according to the manufacturer’s instructions. PCR amplification and HRM analysis were carried out in a LightCycler® 480 real-time PCR system (Roche Diagnostics).\n\nPCR amplification started with an initial denaturation of 95°C for 10 minutes (which included activation of FastStartTM Taq DNA polymerase and denaturation of DNA), followed by 45 cycles of amplification (denaturation at 95°C for 10 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 12 seconds). Melting was performed with a denaturation step at 95°C for 1 minute, followed by an annealing step at 40°C for 1 minute and a melt from 70 to 97°C at a ramp rate of 0.03°C/second with 18 acquisitions per degree Celsius. These results were analyzed using LightCycler® 480 software version 1.5.0.\n\nSanger sequencing was conducted using Applied Biosystems 3500 Genetic Analyzer. For each reaction, 200 ng of genomic DNA was amplified with the following PCR conditions: an initial 10-minute denaturation at 95°C followed by 40 cycles of 30 seconds at 95°C; 30 seconds at 60°C; 30 seconds at 72°C, and a final extension of 7 minutes at 72°C. Sequencing were aligned and edited with the BioEdit Software version 7.2.5.\n\nSamples with conflicting results by HRM and sequencing were re-tested and only HRM-positive samples confirmed by sequencing were considered mutated.\n\nDNA extracted first underwent bisulphite treatment to convert all unmethylated cytosine to uracil, leaving 5-methylcytosine unaltered. After being eluted in DNase-free water, methylation analysis was commenced using real-time methylation-specific PCR (MSP). PCR amplification was carried out in a LightCycler® 480 real-time PCR system (Roche Diagnostics). QuantiFast®Multiplex PCR Kit (QIAGEN) was used for PCR and DNA was amplified utilizing HotStarTaq Plus DNA polymerase which was included in the kit. Primers used were: MGMT Methylated-forward 5’-TTTCGACGTTCGTAGGTTTTCGC-3’, MGMT Methylated reverse 5’-GCACTCTTCCGAAAACGAAACG-3’ and MGMT Unmethylated-forward 5’ TTTGTGTTTTGATGTTTGTAGGTTTTTGT-3’, MGMT Unmethylated-reverse 5’- AACTCCACACTCTTCCAAAAACAAAACA-3’. Cycling conditions were 95°C for 5 min, followed by 42 cycles of amplification (denaturation at 95°C for 30 seconds, annealing at 59°C for 30 seconds, and extension at 72°C for 30 seconds) and 72°C for 5 min. PCR reactions (15 μl) were analyzed on a 2% agarose gel stained with SYBR Safe. Commercial methylated DNA and unmethylated DNA (Zymo Research) served as positive controls. Outcomes were classified as either methylated or unmethylated.\n\nNeurological function was assessed using the NANO scale. NANO scale is a simple neurological assessment evaluating patients in nine domains: gait, strength, upper extremity ataxia, sensation, visual fields, facial strength, language, level of consciousness, and behaviour. Each domain was scored from 0 to 2 or 3, with higher score indicating worse neurologic function. Neurological scale was assessed by two individual physicians, and subjective complaints not included in the scale were also noted. Any discrepancies between investigators were discussed and optimal scale were determined. NANO scale was first assessed before surgery and then re-assessed three months later during their routine clinical follow up. Patients who were deceased before the second assessment were excluded from the study. Neurological improvement was defined as decrement in NANO scale score by 1 or more, which was calculated from difference of initial NANO and NANO three months after surgery.\n\nThe association between baseline characteristics and biomarkers were analysed. Categorical data are presented as proportions and interpreted using chi-square or Pearson Fisher’s exact test. All calculations were performed using IBM SPSS Statistics version 24. The reported p values are two-sided, and a probability value of <0.05 was considered statistically significant.\n\n\nResults\n\nIn total, 54 patients completed the follow up and were analysed. Half of the cases (50%) were glioma grade IV, followed by grade II (37%) and grade III (13%). Overall mean age was 43.63 (14.723) years old for all gliomas, and mean age for glioblastoma was 50 (12.7) years old. Male to female ratio was 1.57. Median Karnofsky Performance Scale (KPS) score was 55 (30–80). Most of the patients underwent surgery followed by radiation and chemotherapy (79.6%) (Table 1).\n\nKPS: Karnofsky Performance Scale; IDH: isocitrate dehydrogenase; WHO: World Health Organisation.\n\nThe most common presenting symptom was headache (63%). Patients with tumours located in the frontal lobe most commonly presented with headache (58.8%), one-sided weakness (35.3%), seizure (32.4%), cognitive disturbance (20.6%), and aphasia (5.9%).\n\nIn 16 patients (29.6%) an IDH1 mutation was found and no IDH2 mutation was found. Younger age and male gender were significantly associated with having an IDH mutation (Table 2).\n\nKPS: Karnofsky Performance Scale; IDH: isocitrate dehydrogenase.\n\nThere were 33 patients (61.1%) that were MGMT methylated (Table 3). Older patients had a higher tendency to have methylated MGMT than younger patients. Patients with IDH mutations were more likely to harbour MGMT methylation (p=0.049; OR=1.54; 95%CI 1.05-2.26).\n\nKPS: Karnofsky Performance Scale; IDH: isocitrate dehydrogenase; MGMT: O6-Methylguanine-DNA methyltransferase.\n\nThe number of patients with MGMT methylation who underwent surgery plus chemoradiation, surgery plus radiation only, and surgery only were 26 patients (78.8%), one patient (3%) and six patients (18.2%), respectively.\n\nMedian NANO scale score before surgery was 4 (0–12) and three months after surgery was 3 (0–12). Improvement in neurological function, measured using the NANO scale, was found in 44 (81.5%) of the patients. Age, gender, initial KPS, tumour location, grade of glioma and IDH mutation were not associated with improvement in neurological function. Among patients with MGMT promoter gene methylation, 90.9% showed improvement in neurological function (p=0.035; OR=5; 95%CI 1.122-22.272) (Table 4).\n\nKPS: Karnofsky Performance Scale; IDH: isocitrate dehydrogenase; MGMT: O6-Methylguanine-DNA methyltransferase; NANO: Neurologic Assessment in Neuro-Oncology.\n\nFurther analysis shows that coexistence of IDH mutation and MGMT methylation were mostly found in grade II patients (53.8%) and 100% of patients with coexistence of both biomarkers showed improvement in neurological scale (p=0.032) (Table 5).\n\nIDH: isocitrate dehydrogenase; MGMT: O6-Methylguanine-DNA methyltransferase; NANO: Neurologic Assessment in Neuro-Oncology.\n\n\nDiscussion\n\nGlioma remains to be a challenging tumour, with diverse clinical presentation, phenotype and molecular parameters (Louis et al., 2016). It is not unusual for clinicians to encounter cases of higher-grade glioma with longer survival than lower-grade glioma during conventional treatment. Despite survival, clinical and functional status is another concern in treatment initiation and disease progression. KPS has been generally used to evaluate brain tumours’ performance status. However, like tumour grade, there have also been cases in which those with a low KPS score survived longer than those with a high KPS score (Lee et al., 2018).\n\nThe NANO scale is a relatively new scale that serve as an objective and quantifiable metric of neurologic function in brain tumour patients (Nayak et al., 2017). A previous study found that performance status estimated by the NANO scale was significantly associated with overall survival, and was a more powerful method to predict the prognosis of GBM than the KPS during both initial diagnosis and disease progression (Lee et al., 2018). It was also found that initial NANO scale possesses a stronger correlation neurological scale improvement than initial KPS towards functional scale improvement (Gunawan et al., 2020). In this study, we used NANO scale to objectively measure neurological scale improvement.\n\nFrom 54 patients included in the study, most of the cases were glioma grade IV, glioblastoma, IDH-wildtype (44.4%), followed by grade II and III. This distribution is similar to previous studies analysing different grades of gliomas (Malueka et al., 2020; Ostrom et al., 2018; Theresia et al., 2020). Mean age in this study was 43.63 years old, and patients with glioblastoma have a mean age of 50 (12.7) years old, which is comparable to previous studies conducted in Asian (Lee et al., 2018; Malueka et al., 2020; Theresia et al., 2020), African (Senhaji et al., 2017), and Amsterdam (Molenaar et al., 2014) populations and lower than mean age of studies in the United Kingdom (Philips et al., 2018; Sehmer et al., 2014), Greece (Alifieris & Trafalis, 2015) and United States (Ostrom et al., 2018; Schwartzbaum et al., 2006). The male to female ratio was 1.57, which is comparable to previous studies (Malueka et al., 2020; Ostrom et al., 2018; Sehmer et al., 2014). The proportion of low and high initial functional status was equivalent. Tumours were mostly located in the frontal lobe, followed by temporal, parietal, and occipital. The most common presenting symptom was headache, and 58.8% of tumours located in the frontal lobe presented with this symptom. Surgery, radiation, and chemotherapy were underwent by 79.6% of patients.\n\nIt was found that 29.6% of patients were positive for IDH1 mutation. Being of a younger age and male were factors associated with having an IDH mutant. This is similar to previous studies that found younger age to be associated with IDH mutation in glioblastoma (Molenaar et al., 2014; Songtao et al., 2012). However, a study in Indonesia showed no differences in age and gender towards IDH mutation (Malueka et al., 2020). Dissimilarity may be caused by differences in characteristics of patients included in their study (which also included grade I glioma). Grade I glioma is frequently found in children, with different characteristics, therapeutic interventions, and prognosis. Therefore, in this study, we did not include grade I glioma and excluded patients less than 10 years old.\n\nMGMT methylation was found in 61.1% of patients. Patients with IDH mutation were more likely to have an MGMT methylation as well (p=0.049; OR=1.54; 95%CI 1.05-2.26). These findings are in accordance with previous studies (Molenaar et al., 2014; Songtao et al., 2012).\n\nImprovement in NANO scale within three months after surgery was found in 81.5% of the patients. From the variables studied, only MGMT promoter gene methylations revealed a significant link to NANO scale improvement three months after surgery (p=0.035; OR=5; 95%CI 1.122-22.272).\n\nAlthough IDH mutation alone was not significantly related to scale improvement, the combination of IDH mutation and MGMT methylation was associated with improvement in neurological function three months later. This might be due to the significant association of MGMT methylation towards NANO scale improvement, which influenced the relation between its combination with IDH mutation towards scale improvement.\n\nMGMT promoter gene methylation has been commonly accepted to confer survival advantage regardless of therapy (Reifenberger et al., 2014). It carries both prognostic and predictive value. In this study, it was found that gliomas with MGMT gene promoter methylation are five times likely to show neurological improvement three months after surgery.\n\nIDH mutation is one of the molecular tests well known for its prognostic and predictive implications in high-grade as well as some low-grade gliomas. Patients with IDH mutation have been shown to convey a longer overall survival and progression-free survival compared to those in the wild-type group (Malueka et al., 2020; Molenaar et al., 2014; Reifenberger et al., 2014; Songtao et al., 2012). However, it is still unclear if IDH mutational status is a prognostic and predictive measure of response to treatment. Some studies have concluded that IDH mutation is an independent factor towards response to treatment (Hartmann et al., 2011; Houillier et al., 2010), while others linked the relation to other markers such as 1p19q deletion (Iwamoto et al., 2008; Jenkins et al., 2006; Mariani et al., 2006) and G-CIMP phenotype (Reifenberger et al., 2014). Our study found no association between IDH mutation and neurological improvement. This might be due to the difference in study outcome, which is improvement in neurological function rather than survival or progression-free survival. Secondly, since we used the difference between initial NANO scale score and score three months later as a measure for neurological improvement, it is possible that patients with low initial scale scores had a persistent score three months later, which is then assessed as not having improvement after three months. Third, the NANO scale as a measure for neurological improvement does not integrate headaches into the scale, which is the most common presenting symptom in this study.\n\nThere are several limitations of this study. First, we did not analyse other predictive biomarkers such as 1p19q deletion and G-CIMP phenotype. Second, the follow up period of three months after surgery might not reveal changes in neurological improvement, since some patients might still be in the chemotherapy and radiation therapy. Hopefully, future studies could integrate other biomarkers as well and conduct a longer period of follow up.\n\n\nConclusions\n\nMGMT promoter gene methylation as well as coexistence of IDH mutation and MGMT methylation shows a significant link to improvement in NANO scale score three months after surgery. Glioma patients with MGMT gene promoter methylation are five times more likely to show neurological improvement three months after surgery.\n\n\nData availability\n\nZenodo: MGMT promoter gene methylation and Neurological Scale Improvement in Glioma. https://doi.org/10.5281/zenodo.4482094 (Gunawan et al., 2021)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nAlifieris C, Trafalis DT: Glioblastoma multiforme: Pathogenesis and treatment. Pharmacol Ther. 2015; 152: 63–82. 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PubMed Abstract \| Publisher Full Text\n\nStupp R, Mason WP, Van Den Bent MJ, et al.: Radiotherapy plus Concomitant and Adjuvant Temozolomide for Glioblastoma. N Engl J Med. 2005; 352(10): 987–96. PubMed Abstract \| Publisher Full Text\n\nTheresia E, Malueka RG, Pranacipta S, et al.: Association between Ki-67 labeling index and histopathological grading of Glioma in Indonesian population. Asian Pac J Cancer Prev. 2020; 21(4): 1063–1068. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "80084", "date": "04 Mar 2021", "name": "Young Zoon Kim", "expertise": [ "Reviewer Expertise 1. Research Interest and Activity 1) Cancer Biology (Invasion", "Metastasis", "Oncogenesis) on malignant brain tumors 2) Cancer Epigenentics (Histone modification) on malignant brain tumors 2. Clinical Practices 1) Neurooncological care for the patients with solid cancer (brain metastasis", "neurocognitive function", "brain tumor related epilepsy) 2) Chemotherapy for brain tumor patients (malignant primary brain tumor", "primary CNS lymphoma", "brain metastasis) 3) Microscopical neurosurgery for brain tumors 4) Neuroendoscopic surgery for skull base tumors" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nFirst of all, I’d like to give a great congratulation to them for nice and successful study. They identified Gliomas with MGMT promoter gene methylation are more likely to show neurological improvement three months after surgery. It is well known for MGMT promoter methylation status to play an important role in predicting prognosis of glioma patients. They showed that MGMT promoter methylation status also have important role in improving the neurological outcome which is estimated by NANO scale. I think that the topic and idea is much novel enough to attract great interest to the readers. Their study was well designed and methods were also reasonable and scientific as well.\nAuthors determined the improvement of neurological status if there was a decrement of ≥1 of the NANO scale. The cut-off value should be validated by use of other neurologically estimating scale such as KPS or WHO performance scale.\n\nReaders may wonder if MGMT methylation status can be independent factor for influencing on NANO scale in glioma patients or not. More statistical analysis such as multivariate analysis using Cox regression model is mandatory to prove their conclusions. Because the patients with methylated MGMT promoter had also IDH-mutation which is favourable prognostic factor in glioma.\n\nAlthough the sample size of glioblastoma patients is small, subgroup analysis focused on glioblastoma patients may be informative to readers.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "6491", "date": "30 Mar 2021", "name": "Pricilla Yani Gunawan", "role": "Author Response", "response": "Dear Mr Young Zoon Kim. I would like to express my gratitude for having you as one of my expert reviewer. It is such an honour to have your detailed and wonderful comments on my manuscript. Here are my responses to your suggestions. 1. The cut-off value of neurological improvement as decrement in NANO scale by 1 or more was made through consideration of the results and definition used in previous studies. I have added the explanation of this subject in the Discussion section, at the end of the second paragraph. 2. Multivariate analysis has been done using a logistic regression analysis to confirm that MGMT methylation status is an independent factor influencing neurological scale improvement. This statement has been added in the Results section, before Table 4. 3. A subgroup analysis focused on glioblastoma patients was added in the Results section, along with Table 6 and Table 7 to further describe the analysis. It was also added in the Discussion section, at the end of the eight paragraph. Thank You for your kind and valuable suggestions. Best Regards, Pricilla et al" } ] }, { "id": "80085", "date": "16 Mar 2021", "name": "Ery Kus Dwianingsih", "expertise": [ "Reviewer Expertise Molecular Pathology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript described MGMT methylation status and IDH1/2 mutation in glioma patients in Indonesian population which showed neurological improvement three months post-surgery.\nThe manuscript is well described, however, there are some rooms for improvement. As we know that the IDH1/2 mutation and MGMT methylation status in glioma patients in asian population have been reported previously in some other publications and its associations with prognosis have also been widely published. The authors may add other highlights in the manuscript to make it more appealing, such as radiological features or other clinical parameters of the subjects or even additional molecular analysis.\n\nHow was the pathological examination performed? What are the criteria for sample to be included in this study? These are not clearly explained in the manuscript.\n\nIn this study the patients were followed for only 3 months post-surgery, which is not long enough to determine the patients' outcome. Longer follow up is needed to give better insight of the MGMT profile related to its prognostic evaluation as seen in previous study conducted by lee et al, in 2018, which was also cited in the manuscript.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6492", "date": "30 Mar 2021", "name": "Pricilla Yani Gunawan", "role": "Author Response", "response": "Dear Mrs. Ery Kus Dwianingsih. First of all, I would like to express my gratitude for having you as one of my expert reviewer. It is a great honour to have your suggestions regarding my manuscript. Below are my responses to your suggestions. 1. IDH mutation and MGMT methylation are unquestionably related with better prognosis in glioma. However, the significance of these biomarkers towards neurological scale improvement has not been extensively studied, especially with the NANO scale. The aim of the study is to pin point the relation between the biomarkers and the short term improvement in neurological scale. 2. The details of how the pathological examination was performed, as well as the criteria for sample to be included in this study was added in the Methods section, under the 'Histopathological and molecular analysis' heading. 3. We agree that the length of follow-up was of short term, hence further studies with longer period of follow-up may give a better insight. For that reason, we have stated this matter under the Discussion section, at the last paragraph. Thank you for your kind and valuable suggestions. Best Regards, Pricilla et al" } ] } ]	1	https://f1000research.com/articles/10-139
https://f1000research.com/articles/10-60/v1	01 Feb 21	{ "type": "Research Article", "title": "Local adaptation in populations of Mycobacterium tuberculosis endemic to the Indian Ocean Rim", "authors": [ "Fabrizio Menardo", "Liliana K. Rutaihwa", "Michaela Zwyer", "Sonia Borrell", "Iñaki Comas", "Emilyn Costa Conceição", "Mireia Coscolla", "Helen Cox", "Moses Joloba", "Horng-Yunn Dou", "Julia Feldmann", "Lukas Fenner", "Janet Fyfe", "Qian Gao", "Darío García de Viedma", "Alberto L. Garcia-Basteiro", "Sebastian M. Gygli", "Jerry Hella", "Hellen Hiza", "Levan Jugheli", "Lujeko Kamwela", "Midori Kato-Maeda", "Qingyun Liu", "Serej D. Ley", "Chloe Loiseau", "Surakameth Mahasirimongkol", "Bijaya Malla", "Prasit Palittapongarnpim", "Niaina Rakotosamimanana", "Voahangy Rasolofo", "Miriam Reinhard", "Klaus Reither", "Mohamed Sasamalo", "Rafael Silva Duarte", "Christophe Sola", "Philip Suffys", "Karla Valeria Batista Lima", "Dorothy Yeboah-Manu", "Christian Beisel", "Daniela Brites", "Sebastien Gagneux", "Liliana K. Rutaihwa", "Michaela Zwyer", "Sonia Borrell", "Iñaki Comas", "Emilyn Costa Conceição", "Mireia Coscolla", "Helen Cox", "Moses Joloba", "Horng-Yunn Dou", "Julia Feldmann", "Lukas Fenner", "Janet Fyfe", "Qian Gao", "Darío García de Viedma", "Alberto L. Garcia-Basteiro", "Sebastian M. Gygli", "Jerry Hella", "Hellen Hiza", "Levan Jugheli", "Lujeko Kamwela", "Midori Kato-Maeda", "Qingyun Liu", "Serej D. Ley", "Chloe Loiseau", "Surakameth Mahasirimongkol", "Bijaya Malla", "Prasit Palittapongarnpim", "Niaina Rakotosamimanana", "Voahangy Rasolofo", "Miriam Reinhard", "Klaus Reither", "Mohamed Sasamalo", "Rafael Silva Duarte", "Christophe Sola", "Philip Suffys", "Karla Valeria Batista Lima", "Dorothy Yeboah-Manu", "Christian Beisel", "Daniela Brites" ], "abstract": "Background: Lineage 1 (L1) and 3 (L3) are two lineages of the Mycobacterium tuberculosis complex (MTBC) causing tuberculosis (TB) in humans. L1 and L3 are prevalent around the rim of the Indian Ocean, the region that accounts for most of the world’s new TB cases. Despite their relevance for this region, L1 and L3 remain understudied. Methods: We analyzed 2,938 L1 and 2,030 L3 whole genome sequences originating from 69 countries. We reconstructed the evolutionary history of these two lineages and identified genes under positive selection. Results: We found a strongly asymmetric pattern of migration from South Asia toward neighboring regions, highlighting the historical role of South Asia in the dispersion of L1 and L3. Moreover, we found that several genes were under positive selection, including genes involved in virulence and resistance to antibiotics . For L1 we identified signatures of local adaptation at the esxH locus, a gene coding for a secreted effector that targets the human endosomal sorting complex, and is included in several vaccine candidates. Conclusions: Our study highlights the importance of genetic diversity in the MTBC, and sheds new light on two of the most important MTBC lineages affecting humans.", "keywords": [ "Mycobacterium tuberculosis", "adaptation", "coevolution" ], "content": "Introduction\n\nThe global tuberculosis (TB) epidemic is a major public health emergency, disproportionately affecting vulnerable populations and worsening existing inequalities. Although the estimated incidence and the number of fatalities have slowly decreased over time, every year ten million people develop the disease, and 1.4 million TB patients die (WHO, 2020). Moreover, the Covid-19 pandemic will likely set back the progress toward TB eradication for several years (Glaziou, 2020; Hogan et al., 2020; McQuaid et al., 2020; Saunders & Evans, 2020). TB is spread worldwide, but not all regions are equally affected: 95% of new TB cases occur in Africa and Asia, and the eight countries with the largest burden account for two thirds of the total number of cases (WHO, 2020).\n\nHuman TB is caused by members of the Mycobacterium tuberculosis complex (MTBC), which includes nine phylogenetic lineages with different geographic distributions. Five of these lineages are restricted to Africa (L5, L6, L7, L8, and L9), while the remaining four (L1, L2, L3, L4) are more broadly distributed (Chihota et al., 2018; Coscollá et al., 2020; Gagneux, 2018; Ngabonziza et al., 2020). While L2 and L4 strains occur across the world, L1 strains are predominantly found around the rim of the Indian Ocean (East Africa, South Asia, and Southeast Asia). L3 has a distinct geographic range (East Africa, Central Asia, Western Asia, and South Asia) that overlaps partially with L1 (Couvin et al., 2019; Wiens et al., 2018). While many MTBC lineages also occur in Northern Europe, North America, Australia and New Zealand, in these low-burden regions, the majority of TB cases are imported through recent migrations, and local transmission is rare (White et al., 2017).\n\nBeyond their geographic distribution, MTBC lineages also differ in virulence, transmissibility, association with drug resistance, and the host immune responses they elicit (Peters et al., 2020). There is an increasing notion that MTBC genetic variation should be considered in the development of new antibiotics and vaccines, and when studying the epidemiology and pathogenesis of the disease (Gagneux, 2017; Peters et al., 2020; Wiens et al., 2018). Yet, much of TB research to date has focused on the laboratory strain H37Rv (belonging to L4) and a few other strains belonging to L2 and L4, because of their broad geographic range, and their association with drug resistance. By contrast, L1 and L3 have largely been neglected, and only few studies have investigated the global populations of these two lineages (Couvin et al., 2019; O’Neill et al., 2019). This knowledge gap is particularly severe as L1 and L3 are endemic to some of the world regions with the heaviest TB burden. For example, L1 and L3 cause the majority of TB cases in India, the country with the highest number of new TB cases in the world, and L1 is by far the most important cause of TB in the Philippines, the country with the 4th highest global TB burden (Wiens et al., 2018). The aim of this study was to characterize the global population structure of L1 and L3 using large-scale population genomics, and to investigate the evolutionary history and selective forces acting on these two lineages.\n\n\nResults\n\nWe screened a large collection of publicly available MTBC genome sequences and selected those belonging to L1 and L3. Additionally, we newly sequenced 767 strains to cover regions that were not well represented in the public data. We applied a series of bioinformatic filters to exclude low quality sequences and mixed infections (Methods), and obtained a curated genome dataset of 4,968 strains (2,938 L1, 2,030 L3). While the phylogenetic tree of L3 showed a ladder-like topology without an evident division in sublineages, L1 comprised five clearly distinct sublineages coalescing close to the root of the tree (Extended data: Figures 1 and 2).\n\na) Heatmap indicating the origin of the 2,061 L1 strains included in the dataset used for the biogeography analysis. b) The geographic regions used in the biogeography analysis of L1. c) Results of PASTML (compressed tree), the color code follow the legend of panel (b), the size of the circles is proportional to the number of tips, and the size of the arrows is proportional to the number of times the pattern of migration was observed on the tree. This plot excluded nodes that represented less than three strains. The same plot including all nodes can be found as Extended Data File 1. d) Tree obtained with the Mascot analysis; the branches are colored following the legend in panel (b) and represent the inferred ancestral region with the largest posterior probability. e) Representation of the relative effective population sizes (circles) and migration rates (lines connecting circles) estimated by Mascot. Lines representing migration rates are colored based on the region of origin (interpreted forward in time). For example, the dark blue line connecting the dark blue circle with the red circle represents the forward migration rate between South Asia and East Africa, which is the largest migration rate estimated by Mascot. The parameter estimates are reported in Extended Data Table 2.\n\na) Heatmap indicating the origin of 1,021 L3 strains included in the dataset used for the biogeography analysis. b) The geographic regions used in the biogeography analysis of L3. c) Results of PASTML (compressed tree), the color code follows the legend of panel (b), the size of the circles is proportional to the number of tips, and the size of the arrows is proportional to the number of times the pattern of migration was observed on the tree. This plot excluded nodes that represent less than three strains, the same plot including all nodes can be found as Extended Data File 3. d) Tree obtained with the Mascot analysis; the branches are colored following the legend in panel (b) and represent the inferred ancestral region with the largest posterior probability. e) Representation of the relative effective population sizes (circles) and migration rates (lines connecting circles) estimated by Mascot. Lines representing migration rates are colored based on the region of origin (interpreted forward in time). The effective population size of South Asia was estimated to be much larger than the one of East Africa. Additionally, the forward migration rate between South Asia and East Africa was much larger than the one in the opposite direction. The parameter estimates are reported in Extended Data Table 2.\n\nThe genome sequences included in our final dataset represented 69 countries and covered the known geographic range of L1 and L3 (Methods; Extended data: Table 1 and Figures 3 and 4). Because we were interested in studying L1 and L3 in their endemic range, we excluded strains originating from North America, Europe, and Australia from the biogeographic analysis, as in these low-burden regions, most TB patients are recent migrants who were infected in their country of origin (White et al., 2017). We assigned strains to different geographic regions following a modified version of the United Nation geographic scheme (Methods, Figure 1 and Figure 2). Mapping the regions of origin onto the phylogenetic trees revealed that two sublineages of L1 are found almost exclusively in Southeast Asia (L1.1.1 and L1.2.1), while the others are spread over the complete geographic range of L1 (Figure 1; Extended Data: Figures 5–6).\n\nWe performed a phylogeographic analysis using two alternative approaches (Mascot and PASTML), which are based on different models and inference methods (Ishikawa et al., 2019; Müller et al., 2018). We found that South Asia was predicted to be the geographic range of the Most Recent Common Ancestor (MRCA) of both L1 and L3 (Extended Data: Information). Most interestingly, we found a strongly asymmetric pattern of migration. For L1, PASTML identified most migration events from South Asia toward other regions, followed by further dispersion, but almost no back migration to South Asia (Figure 1; Extended data: Figure 7 and Files 1 and 2). When we estimated the migration rates with Mascot, we found that the forward migration rate from South Asia toward the rest of the world were 3 to 17 times larger than the migration rates in the opposite direction, confirming the results of PASTML (Figure 1; Extended data: Table 2). We found a similar scenario for L3: PASTML inferred the largest number of migrations from South Asia toward East Africa, further spread from East Africa toward neighboring regions, but essentially no migration back to South Asia (Figure 2; Extended data: Files 3–4). The Mascot analysis showed that the forward migration rates from South Asia toward East Africa were 26 times larger than the migration rates in the opposite direction (Figure 2; Extended data: Table 2).\n\nWe performed tip dating to estimate the age of the trees but got inconsistent results for L1, due to a lack of a reliable temporal signal (Extended data: Information, Figures 8 and 9, Tables 3 and 4). However, the results of a previous study suggested a relatively fast evolutionary rate for L1 (~ 1.4×10-7 nucleotide changes per site per year; Menardo et al., 2019), and that the MRCA of L1 lived around the 12th century AD (Extended data: Information). By contrast, L3 had a good temporal signal, and different methods estimated that its MRCA lived between the 2nd and the 13th century AD. However, the uncertainty around all of these estimates was very large (Extended data: Information, Figure 8, Tables 5 and 6). Together, these results corroborate the findings of previous studies (Bos et al., 2014; Duchêne et al., 2016; Menardo et al., 2019). Calibrating MTBC trees that are hundreds or thousands of years old, with sequences sampled in the last few decades is notoriously challenging and subject to limitations (Menardo et al., 2019). Therefore, we refrain from any strong interpretation of the results of the molecular clock analyses of L1 and L3. We emphasize that the ages reported here are the most likely estimates supported by the available data. Additional data, or alternative methods, might result in different temporal scenarios.\n\nWith respect to the geographical aspects, we identified several interesting instances of long-range dispersal. First, we found a clade of L1, composed of 11 strains sampled in five different West African countries. This was surprising because West Africa is usually not considered part of the geographic range of L1. This clade is nested within sublineage L1.1.1, which is essentially only found in mainland Southeast Asia, and the PASTML analysis inferred a direct introduction from Southeast Asia to West Africa. This introduction is unlikely to have happened before the 16th century, when the Portuguese established the maritime route between Europe and Asia by circumnavigating Africa. Assuming that this migration did not occur before the 16th century, we benchmarked the molecular clock of L1 and found that this scenario is indeed compatible with a clock rate equal to, or larger than 1.4×10-7 nucleotide changes per site per year, but not with lower ones (Extended data: Information, Figure 10).\n\nSecond, we found that L1 was introduced to South America on at least 11 independent occasions (Figure 1; Extended data: Files 1 and 2). Assuming a clock rate of 1.4×10-7, the earliest introduction was between 1620 and 1830 AD from East Africa, while subsequent introductions occurred from East Africa, South Africa, and South Asia. These results support the hypothesis that L1 was first introduced to Brazil through the slave trade from East Africa (Allen, 2013; Conceição et al., 2019). Interestingly, this is in contrast with L5 and L6, which are endemic to West Africa, but did not establish themselves firmly in South America (De Jong et al., 2010; Rabahi et al., 2020).\n\nThird, similar to the West African clade of L1.1.1, we found an East African clade embedded within sublineage L1.2.1, which otherwise is found almost exclusively in Southeast Asia. This East African clade is composed of 11 strains from five countries, and its sister clade is found in East Timor and Papua New Guinea. We inferred a direct migration from the islands of Southeast Asia to East Africa that occurred between the 13th and the 16th century AD (assuming a clock rate of 1.4×10-7). This would be compatible with early Portuguese expeditions, which reached East Timor and Papua New Guinea in the early 16th century. An alternative explanation could be the Austronesian expansion. Austronesians are thought to have reached the Comoros islands and Madagascar between the 9th and the 13th century AD, possibly through direct navigation from Southeast Asian islands. Malagasy speak an Austronesian language, and Austronesian genetic signatures are found in human populations in the Comoros, Madagascar, and to a small extent also in the Horn of Africa (Blench, 2010; Boivin et al., 2013; Brucato et al., 2016; Brucato et al., 2018; Brucato et al., 2019; Pierron et al., 2014).\n\nL1 and L3 coexist in many regions around the Indian Ocean. Yet, in their evolutionary history, these lineages colonized areas occupied by different human populations. Human genetic variation has been shown to influence the susceptibility to TB (Qu et al., 2011). Most notably, HLA genes play a crucial role in the activation of the immune responses to the MTBC by exposing bacterial peptides (epitopes) to the surface of an infected cell, where they can be recognized by T cells. HLA genes are extremely polymorphic in human populations, and several alleles of different HLA genes are associated with TB susceptibility in different populations (Brahmajothi et al., 1991; Salie et al., 2014; Sveinbjornsson et al., 2016; Vejbaesya et al., 2002; Yuliwulandari et al., 2010).\n\nPrevious studies have shown that T cell epitopes are hyper-conserved in the MTBC, suggesting that immune escape does not provide an advantage and that, contrary to other pathogens, the MTBC needs to be recognized by the immune system and to cause disease in order to transmit (Comas et al., 2010; Coscolla et al., 2015). Our large dataset of L1 and L3 genome sequences from different geographic regions provided an opportunity to scan for lineage- and/or region-specific signatures of selection at T cell epitopes in L1 and L3.\n\nWe reconstructed the mutational history of T cell epitopes in L1 and L3, and found that 51% of all epitopes were variable (at the amino acid level) in at least one L1 strain, while only 20% were variable in at least one L3 strain (Extended data: Table 7). However, this difference can be explained by the different size and diversity of the two datasets (2,061 genome sequences with 136,023 variable sites for L1; 1,021 genome sequences with 36,316 variable sites for L3). The epitope that accumulated most mutations was located at the N terminus of esxH (Rv0288). This peptide is a T cell epitope for both classes, MHCI and MHCII; it is also a B cell epitope and was previously identified as one of the few T cell epitopes that were not hyper-conserved (Comas et al., 2010). We found 15 derived haplotypes (at the amino acid level), generated by 28 independent replacements at five positions in a peptide of seven amino acids (Figure 3). By contrast, the second most variable epitope accumulated only seven amino acid changes (Extended data: Table 7). Interestingly, we did not find this signature in L3, as all strains carried the ancestral haplotype at the N-terminal esxH epitope. Moreover, when we extended the analysis to two large genomic datasets of L2 and L4 strains, we found a much weaker signal: while 21% of L1 strains carried a derived haplotype for this epitope, only 1% of L2 and L4 strains, and no L3 strain had a derived haplotype. Despite analyzing datasets with more strains (6,752 and 10,466 for L2 and L4, respectively), and more polymorphic positions (140,309 and 277,648) compared to L1, we found only three amino acid replacements at the N-terminal epitope of esxH in L2, and seven in L4 (Figure 3). The most frequently mutated position was the tenth amino acid, where we found 12 independent replacements in L1, and two in L2 and L4 (Figure 3). The amino acid replacement A10V occurred eight times in parallel in L1, once in L2 and twice in L4. The most abundant derived haplotype was caused by a different amino acid replacement at position ten (A10T), which occurred once in L1 and once in L2 (Figure 3). Overall, the replacements in all lineages occurred in eight residues in a peptide of 13 amino acids (Figure 3).\n\nThe ancestral epitope is reported in top position, the derived haplotypes are reported below: mutations that were present in more than one lineage are highlighted in yellow, haplotypes that were found exclusively in L1, L2 or L4 are highlighted in pink, blue and red, respectively. Asterisks indicate the position inferred to be under positive selection by PAML: * posterior probability > 0.95: ** posterior probability > 0.99. The table on the right reports for each lineage the number of strains harboring the corresponding haplotype, and between parentheses, the number of independent parallel occurrences of the mutations as inferred by PAUP.\n\nWe evaluated the robustness of these results by formally testing for positive selection on the complete sequence of esxH using PAML (Yang, 2007). We found that esxH was indeed under positive selection in L1 (p-value = 0.00004) but not in the other lineages (p-values = 0.39, 1.00 and 0.65 for L2, L3 and L4, respectively). PAML identified four codons that have been under positive selection in L1 (posterior probability > 0.95), all of them within the N-terminal epitope (codons 7, 9, 10 and 13; Figure 3). Codon 76, which is part of a different T cell epitope, had a posterior probability > 0.9, mutated three times in parallel and was possibly also under positive selection.\n\nOur results further revealed that the derived haplotypes of the N-terminal esxH epitope were not distributed randomly across the geographic range of L1. Twenty-two of the 28 (79%) amino acid replacements occurred in sublineages L1.1.1 and L1.2.1, which are almost exclusively present in Southeast Asia (Extended data: Figure 11). We constructed a statistical test of association similar to phyC (Farhat et al., 2013) to determine whether replacements in the hypervariable esxH epitope were significantly associated with a particular geographic region (Methods). We found that South African strains were less likely to harbor a derived haplotype in the N-terminal esxH epitope than expected by chance (empirical p-value = 0.013; Table 1). While East African L1 strains were not associated with the derived haplotypes (empirical p-value = 0.276), we noticed important differences between countries: of the 29 East African strains harboring a derived haplotype, 28 were sampled in Madagascar. When we excluded Madagascar, we found that East Africa had a strong negative association with the derived haplotypes (i.e. East African strains harbored less derived haplotypes than expected by chance; empirical p-value = 0.0004). We then tested the most frequently replaced position (position ten) alone, and again found that East African strains were negatively associated with the derived haplotypes, with and without excluding Malagasy strains (empirical p-values = 0.0176 and 0.034, respectively). Finally, we tested the derived haplotype caused by the most frequent amino acid replacement (A10V; 8 parallel occurrences). Again, we found a negative association with East African strains (empirical p-values = 0.046 and 0.079, respectively including and excluding Malagasy strains; Table 1).\n\n1 We included only regions for which the sample size was 25 or more.\n\n2 Total number of L1 strains from each region.\n\n3 All p-values are one-tailed empirical p-values. This column indicates the direction of the association: + we tested for positive association between the derived haplotype(s) and the geographic region; - we tested for negative association between derived haplotype(s) and the geographic region.\n\n4 Number of strains with the derived haplotype caused by the replacement A10V, and results of the test of association. P-values < than 0.1 are shown in bold.\n\n5 Number of strains with the derived haplotypes caused by any replacement at position 10 (A10X), and results of the test of association. P-values < than 0.1 are shown in bold.\n\n6 Number of strains with the derived haplotypes caused by any mutations occurred in the first 18 amino acids of EsxH, and results of the test of association. P-values < than 0.1 are shown in bold.\n\nWe hypothesized that the accumulation of missense mutations at the N-terminal epitope of esxH was due to immune escape. Therefore, we performed in silico prediction of the binding affinity of the ancestral haplotype and of the two most frequent derived haplotypes (caused by the amino acid replacements A10V and A10T) with different HLA-A, HLA-B and HLA-DRB1 alleles (Methods). We performed this analysis for:\n\n1) Alleles found at high frequency (> 10%) in South- and Southeast Asia, but not in East Africa.\n\n2) Alleles found at high frequency (> 10%) in East Africa, but not in South- and Southeast Asia.\n\n3) Alleles found at high frequency (> 10%) in both regions.\n\nHowever, we found no differences in the predicted binding affinities between alleles with different geographic distributions (Extended data: Table 8).\n\nWhile esxH was the most striking example of a selective pressure specific for one lineage, our analysis suggests that it was not an isolated case. We performed a genome-wide scan for selection with PAML, and identified 17 genes under positive selection, of which five in common between L1 and L3 (Extended data: Table 9). We found two genes coding for transmembrane proteins, members of the Esx-1 secretion system, which were under positive selection in L1 (eccB1 and eccCa1, Bonferroni corrected p-values = 0.02 and 0.03), and several genes involved in antibiotic resistance that were under positive selection in both lineages (Extended data: Information). We further characterized the profile of drug resistance mutations of L1 and L3, and found that L1 strains harbored a greater proportion of inhA promoter mutations (conferring resistance to isoniazid) compared to L3 strains, confirming previous findings (Fenner et al., 2012; Extended data: Information, Figure 12, Table 10).\n\n\nDiscussion\n\nOur results highlight the central role of South Asia in the dispersion of L1 and L3. First, we confirmed that the two lineages probably expanded from South Asia (O’Neill et al., 2019). Second, contrary to previous studies that assumed symmetric migration rates between regions (O’Neill et al., 2019), we found that these migrations occurred mostly in one direction. South Asia was a source of migrant strains that fueled the epidemics in other regions, especially in East Africa. Historically, a network of maritime trade, which followed the seasonality of the Monsoons, connected East Africa and South Asia. It is unclear how this could have promoted the spread of TB in one direction, but not in the other. A possible explanation is that strains originating in South Asia were more efficient in transmitting in East Africa, compared to East African strains that migrated to South Asia.\n\nAnother difference compared to previous studies is the temporal framework for the evolutionary history of L1. O’Neill et al. (2019) estimated that the MRCA of L1 lived in the 4th century BC and that the migration rates decreased after the 7th century AD. Our results indicate that the MRCA of L1 did not exist before the 12th century AD. This discrepancy is due to different assumptions about the clock rates of L1, but none of the two hypotheses can be excluded with the available data. Nonetheless, our discovery of a West African clade that originated through a direct introduction from Southeast Asia supports a faster clock rate of L1, and therefore a more recent MRCA (Extended data: Information). As we already mentioned, tip-dating analyses of MTBC trees with roots that are several hundreds of years old is extremely challenging, and the results of such analyses should be taken with caution (Menardo et al., 2019). Moreover, all MTBC molecular clock studies so far assumed that evolutionary rate estimates do not depend on the age of the calibration points (Ho et al., 2005). There are some indications that the effect of time dependency in MTBC datasets is negligible (Pepperell et al., 2013; Menardo et al., 2019). However, this assumption has not yet been thoroughly tested due to the lack of appropriate samples.\n\nWe found evidence for a strong selective pressure acting on the N terminus of esxH in L1. In contrast, this selective pressure seems to be much reduced, if not absent, in the “modern” lineages (L2, L3 and L4). It is known that L1 strains interact differently with the infected hosts compared to other lineages. For example, L1 strains show a lower virulence in animal models (Bottai et al., 2020), transmit less efficiently in some clinical settings, and infect older patients (Holt et al., 2018). It has been shown recently that the increased virulence of the so-called “modern” MTBC strains is due to the loss of the genomic region TbD1, which remains present in L1 (Bottai et al., 2020). However, it was also reported that in some populations, L1 was associated with higher patient mortality (Smittipat et al., 2019). Given these differences with the “modern” lineages, it is likely that L1 is subject to different selective pressures, and it is possible that the greater selective pressure on esxH was caused by some epistatic effect specific to L1.\n\nThe signatures of selection at the N-terminal epitope of esxH were associated with strains sampled in South- and Southeast Asia, and were almost completely absent in East Africa (excluding Madagascar). Region-specific signatures of positive selection are a hallmark of local adaptation; in this case, adaptation of L1 strains to human hosts with South- and Southeast Asian genotypes. This corroborates previous studies reporting that in Taiwan, L1 is associated with indigenous populations with Austronesian ancestry (reviewed in Dou et al., 2015). This hypothesis is also supported by the L1 population in Madagascar. Madagascar is geographically linked to East Africa, however, Malagasies are genetically distinct from Africans, as they have mixed African and Southeast Asian ancestry due to the Austronesian colonization of Madagascar (Brucato et al., 2016). Madagascar was the region with the second highest prevalence of derived haplotypes in the N-terminal epitope of esxH after Southeast Asia (islands), 37.3% and 72.8% respectively, opposed to 0.3% (one single strain) in the rest of East Africa.\n\nAlthough all the codons under positive selection in esxH were contained in one single T cell epitope, the selective pressure acting on esxH could be due to some other factor, and not to the binding of the epitope by the MHC, or to its recognition by T cells. A possible mechanism was suggested by experiments in a mouse model, showing that the N-terminal epitope of EsxH generates an immunodominant CD8 T cells response. The amino acid replacement A10T (which we found to be the most prevalent among the derived haplotypes of the N-terminal EsxH epitope) did not change MHC binding or T cell recognition, but it accelerated the degradation of the epitope, disrupting the immunodominant response (Sutiwisesak et al., 2020; Woodworth et al., 2008).\n\nAn additional driver of selection could be the effector function of EsxH. EsxH is a small effector secreted by the Esx-3 secretion system as dimer with EsxG (Ilghari et al., 2011). Within the host macrophages, the dimer EsxH-EsxG targets Hrs (a component of the human endosomal sorting complex), impairing trafficking, and hindering phagosomal maturation and antigen presentation, thus contributing to the survival of the pathogen (Mehra et al., 2013; Mittal et al., 2018; Portal-Celhay et al., 2016). The observed signatures of selection could be due to the adaptation of L1 strains to human genotypes of hrs prevalent in South- and Southeast Asia. A similar signature of selection was observed in another Esx effector (EsxW), in MTBC strains belonging to L2 (Holt et al., 2018). Holt and colleagues found evidence for parallel evolution at one residue in the N-terminal loop of EsxW, outside the region covered by known epitopes, suggesting that the selective pressure on EsxW was not due to antigen recognition.\n\nThe sampling effort in this study was considerable, and it provided a more complete picture compared to previous studies. Nevertheless, the sampling was not population-based, and for some regions the coverage was scarce (e.g. the Arabian Peninsula). Because of these limitations, our biogeographical analyses were limited to the subcontinental level. This approach revealed the global population structure and the main macroscopic patterns of diversity and migration of L1 and L3. However, MTBC populations are diverse also within subcontinental regions. For example, the MTBC population in Southern India is dominated by L1, while the most prevalent lineage in the North is L3 (Couvin et al., 2019). To investigate fine-scale processes in greater detail, including local adaptation, large population-based studies will be necessary.\n\nIn conclusion, the results presented here improve our knowledge about the TB epidemic around the Indian Ocean. A better understanding of the evolutionary dynamics of different MTBC populations might inform the development of control strategies for different regions. For example, esxH is part of several new vaccine candidates (Abel et al., 2010; Hoang et al., 2013; Radošević et al., 2007), and at least one of these, H4:IC31, is under clinical development in South Africa (Bekker et al., 2020; Nemes et al., 2018). In the light of our findings, and to develop a globally effective vaccine, it would be important to know if the results of the clinical trials in South Africa can be replicated in Southeast Asia, where there is a high prevalence of derived esxH haplotypes in the circulating MTBC populations.\n\n\nMethods\n\nMTBC isolates previously identified as L1 and L3, either by SNP-typing or spoligotyping, were grown in Middlebrook 7H9 liquid medium supplemented with ADC and incubated at 37°C. Purified genomic DNA was obtained from cultures using a CTAB extraction method. Whole genome sequencing was performed on libraries prepared from purified genomic DNA using Illumina Nextera ® XT library and NEBNext ® Ultra TM II FS DNA Library Prep Kits. Sequencing was performed using the Illumina HiSeq 2500 or NextSeq 500 paired-end technology.\n\nThe sequence data generated by this study has been deposited on SRA under the accession numbers PRJNA630228 and PRJNA670836.\n\nWe screened a large collection of publicly available whole genome sequences (Illumina) of MTBC strains belonging to L1 and L3, using the diagnostic SNPs described in Steiner et al. (2014). To cover geographic regions that were under-represented by this dataset, we additionally sequenced the genomes of 767 clinical strains (360 L1, and 407 L3).\n\nFor all samples, Illumina reads were trimmed with Trimmomatic v0.33 (SLIDINGWINDOW: 5:20,ILLUMINACLIP:{adapter}:2:30:10) (Bolger et al., 2014). Reads shorter than 20 bp were excluded from the downstream analysis. Overlapping paired-end reads were then merged with SeqPrep (overlap size = 15; https://github.com/jstjohn/SeqPrep). The resulting reads were mapped to the reconstructed MTBC ancestral sequence (Comas et al., 2013) with BWA v0.7.12 (mem algorithm; Li & Durbin, 2010). Duplicated reads were marked by the MarkDuplicates module of Picard v 2.1.1 (https://github.com/broadinstitute/picard). The RealignerTargetCreator and IndelRealigner modules of GATK v.3.4.0 (McKenna et al., 2010) were used to perform local realignment of reads around Indels. Reads with alignment score lower than (0.93read_length)-(read_length40.07)) were excluded: this corresponds to more than 7 miss-matches per 100 bp.\n\nSNPs were called with Samtools v1.2 mpileup (Li et al., 2009) and VarScan v2.4.1 (Koboldt et al., 2012) using the following thresholds: minimum mapping quality of 20, minimum base quality at a position of 20, minimum read depth at a position of 7X, minimum percentage of reads supporting the call 90%. SNPs in previously defined repetitive regions were excluded (i.e. PPE and PE-PGRS genes, phages, insertion sequences and repeats longer than 50 bp) as described before (Brites et al., 2018).\n\nWe applied the following filters: genomes were excluded if they had 1) an average coverage <15x, 2) more than 50% of their SNPs excluded due to the strand bias filter, 3) more than 50% (or more than 1,000 in absolute number) of their SNPs having a percentage of reads supporting the call between 10% and 90%, 4) contained single nucleotide polymorphisms diagnostic for different MTBC lineages (Steiner et al., 2014), as this indicated that a mix of genomes was sequenced, 5) had more than 5,000 SNPs of difference compared to the reconstructed ancestral genome of the MTBC (Comas et al., 2013). Additionally, when multiple strains were sampled from the same patient, we retained only one. We further excluded all strains that had less SNPs than (average - (3 standard deviation)) of the respective lineage (calculated after all previous filtering steps). We built SNP alignments for L1 and L3 separately, including only variable positions with less than 10% of missing data, and finally, we excluded all genomes with more than 10% of missing data in the alignment of the respective lineage. After all filtering steps, we were able to retrieve 4,968 strains with high quality genome sequences for further analyses (2,938 L1, 2,030 L3; Extended data: Table 1).\n\nWe used the curated datasets and inferred phylogenetic trees based on all polymorphic positions (excluding the ones in repetitive regions, see above) with raxml 8.2.11 (Stamatakis, 2014; -m GTRCAT and -V options). We then identified sublineages following the classification (and using the diagnostic SNPs) of Coll et al. (2014).\n\nWe selected all strains for which the year of sampling was known (2,499 strains, 1,672 L1, 827 L3). For both lineages, we built SNP alignments including only variable positions with less than 10% of missing data. We inferred phylogenetic trees with RAxML 8.2.11 (Stamatakis, 2014), using the GTR model (-m GTRCAT -V options). Since the alignments contain only variable positions, we rescaled the branch lengths of the trees: rescaled_branch_length = ((branch_length * alignment_lengths) / (alignment_length + invariant_sites)). We rooted the trees using the genome sequence of a L2 strain as outgroup (accession number SAMEA4441446).\n\nWe used the least square method implemented in LSD v0.3-beta (To et al., 2016) to estimate the molecular clock rate with the QPD algorithm and calculating the confidence interval (options -f 100 and -s). We also performed a date randomization test by randomly reassigning the sampling dates among taxa 100 times and estimating the clock rate from the randomized and observed datasets. All LSD analyses were performed on the two lineages individually (L1 and L3), and on the five sublineages of L1.\n\nBayesian molecular clock analyses are computationally demanding and they would be impossible to apply onto the complete datasets. Therefore, we sub-sampled the L1 and L3 datasets used for the LSD analysis to 400 genomes with two different strategies: 1) random subsampling 2) random subsampling keeping at least 25 genomes for each year of sampling (where possible; “weighted subsampling”). For this second subsampling strategy, we used Treemmer v0.3 (Menardo et al., 2018) with options -X 400, -pr, -lm, and -mc 25. For both subsampling schemes, we generated three subsets of the original datasets, resulting in six subsets for each lineage. We assembled SNP alignments including only variable positions with less than 10% of missing data, and used jModelTest 2.1.10 v20160303 (Darriba et al., 2012) to identify the best fitting nucleotide substitution model (according to the Akaike information criterion) among 11 possible schemes including unequal nucleotide frequencies (total models = 22, options -s 11 and -f).\n\nWe performed Bayesian inference with Beast 2.5 (Bouckaert et al., 2019). We corrected the xml files to specify the number of invariant sites as indicated here: https://groups.google.com/forum/#!topic/beast-users/QfBHMOqImFE, and used the tip sampling year as calibration. We assumed the best fitting nucleotide substitution model as identified by jModelTest, a relaxed lognormal clock model (Drummond et al., 2006) and an exponential population size coalescent prior. We chose a 1/x prior for the population size [0–109], a 1/x prior for the mean of the lognormal distribution of the clock rate [10−10–10−5], a normal(0,1) prior for the standard deviation of the lognormal distribution of the clock rate [0 –infinity]. For the exponential growth rate prior, we used the standard Laplace distribution [-infinity–infinity]. For all datasets, we ran at least two runs, we used Tracer 1.7.1 (Rambaut et al., 2018) to identify and exclude the burn-in, to evaluate convergence among runs and to calculate the estimated sample size. We stopped the runs when at least two chains reached convergence, and the ESS of the posterior and of all parameters were larger than 200.\n\nSince we detected a strong temporal signal only for L3, we performed a set of additional analyses of the subsets of L3. We repeated the Beast analysis with an extended Bayesian Skyline Plot (BSP) prior instead of the exponential growth prior, and performed a nested sampling analysis (Russel et al., 2019) to identify which of these two models (exponential growth and extended BSP) fitted the data best. The nested sampling was run with chainLength = 20000, particleCount= 4, and subChainLength = 10000.\n\nAll xml input files are available as Supplementary Files in Extended data.\n\nFor the biogeography analysis, we considered only genome sequences obtained from strains for which the locality of sampling was known. When the country of sampling did not correspond to the country of origin of the patient (or was unknown), we considered as sampling locality the country of origin of the patient (this affected 187 strains, 121 L1 and 66 L3). Furthermore, similarly to other studies (O’Neill et al., 2019), we excluded all genomes that were sampled from Europe, North America and Australia, as most contemporary infections in these regions affect recent migrants. The final dataset for the biogeography analyses comprised 3,082 strains (2,061 L1 and 1,021 L3). We assigned the different isolates to different subcontinental geographic regions according to sampling locality. To do this, we followed a modified version of the United Nations geographic scheme (https://unstats.un.org/unsd/methodology/m49/; Extended data: Table 1 and Figures 1–3).\n\nFor both lineages, we built SNP alignments including only variable positions with less than 10% of missing data, and all strains with known sampling locality (excluding strains from North America, Europe and Australia; see above, 2,061 L1 genomes and 1,021 L3 genomes). We inferred phylogenetic trees with raxml 8.2.11 (Stamatakis, 2014), using the GTR model (-m GTRCAT -V options). Since the alignments contain only variable positions, we re-scaled the branch lengths of the trees: rescaled_branch_length = ((branch_length * alignment_lengths) / (alignment_length + invariant_sites)).\n\nSince PASTML needs a time tree as input, we calibrated the phylogenies with LSD, assuming a clock rate of 1.4×10-7 for L1, and 9×10-8 for L3. In this analysis, genomes for which the sampling date was not known were assumed to have been sampled between 1995 and 2018, which is the period in which all strains with known date of isolation were sampled. Importantly, using different clock rates for this analysis would only change the time scale of the trees, but not the reconstruction of the ancestral characters.\n\nWe assigned to each strain the subcontinental geographic region of origin as character, and used PASTML (Ishikawa et al., 2019) to reconstruct the ancestral geographical ranges and their changes along the trees of L1 and L3. We used the MPPA as prediction method (standard settings) and added the character predicted by the joint reconstruction even if it was not selected by the Brier score (option -forced_joint). Additionally, we repeated the PASTML analysis for the sublineages of L1 individually.\n\nAs a complementary method to reconstruct the ancestral range and the migration pattern of different populations, we used the Beast package Mascot (Müller et al., 2018). We assumed that strains sampled in the different subcontinental regions represent distinct subpopulations, and we considered only populations for which we had at least 75 genome sequences: four populations for L1 (East Africa, South Asia, Southeast Asia (mainland) and Southeast Asia (islands)), and two populations for L3 (East Africa and South Asia). For computational reasons, we subsampled the two datasets (L1 and L3) to ~300 strains. We sampled an equal number of strains from each geographic region (where possible), and within regions, we randomly sampled an equal number of strains from each country (where possible). This sub-sampling scheme resulted in two subsets of 303 and 300 strains for L1 and L3, respectively.\n\nWe assembled SNP alignments including only variable positions with less than 10% of missing data, and used jModelTest 2.1.10 v20160303 (Darriba et al., 2012) to identify the best fitting nucleotide substitution model as described above.\n\nWe performed Bayesian inference with Beast2.5 (Bouckaert et al., 2019). We corrected the xml files to specify the number of invariant sites as indicated here: https://groups.google.com/forum/#!topic/beast-users/QfBHMOqImFE. We assumed a lognormal uncorrelated clock and we fixed the mean of the lognormal distribution of the clock rate to 1.4×10-7 (L1) and 9×10-8 (L3). We assigned the tip sampling years to the different strains, and when the sampling time was unknown, we assumed a uniform prior from 1995 to 2018 (similarly to what done in the PASTML analysis). We further assumed the best fitting nucleotide substitution model as identified by jModelTest, a gamma prior for the standard deviation of the lognormal distribution of the clock rate [0 –infinity], and a lognormal prior for the population size with standard deviation = 0.2, and mean estimated in real space. Finally, we used an exponential distribution with mean = 10-4 as prior for the migration rates. For each analysis, we ran at least two runs. We used Tracer 1.7.1 (Rambaut et al., 2018) to identify and exclude the burn-in, to evaluate convergence among runs, and to calculate the estimated sample size. We stopped the runs when at least two chains reached convergence, and the ESS of the posterior, prior and of the parameters of interest (population sizes and migration rates) were larger than 200.\n\nThe xml input files are available as Supplementary Files in Extended data.\n\nFor the analysis of esxH, we wanted to compare the results obtained for L1 and L3 with the other two major human-adapted MTBC lineages L2 and L4. Therefore, we compiled two datasets of publicly available genome sequences for these two lineages. We applied the same bioinformatic pipeline described above: genomes were excluded if they had 1) an average coverage <15x, 2) more than 50% (or more than 1,000 in absolute number) of their SNPs having a percentage of reads supporting the call between 10% and 90%, or 3) contained single nucleotide polymorphisms diagnostic for different MTBC lineages (Steiner et al., 2014), as this could indicate mixed infection. The final dataset consisted of 6,752 L2 genome sequences (with 140,309 polymorphic positions) and 10,466 L4 genome sequences (with 277,648 polymorphic positions; Extended data: Table 1). We reconstructed the phylogenetic tree of L2 with raxml 8.2.11 (Stamatakis, 2014) as described above. Due to the large size of the dataset, we used FastTree (Price et al., 2010) with options -nt -nocat -nosupport to reconstruct the phylogenetic tree of L4.\n\nWe downloaded the amino acid sequence of all MTBC epitopes described for Homo sapiens from the immune epitope database (https://www.iedb.org/; downloaded on the 03.08.2020). We considered separately MHCI epitopes and MHCII epitopes. We mapped the epitope sequences onto the H37Rv genome (GCF_000195955.2) using tblastn and excluded sequences mapping equally well to multiple loci in the H37Rv genome. Additionally, we retained only epitopes that mapped with two mismatches or less over the whole epitope length. This resulted in a final list of 539 MHCI epitopes, and 1,144 MHCII epitopes. (Extended data: Table 7). We used the datasets obtained for the biogeography analysis (2,061 genome sequences for L1, and 1,021 genome sequences for L3).\n\nFor each lineage, we independently assembled a multiple sequence alignment for each epitope. We then translated the sequence to amino acids and used PAUP 4.a (Wilgenbusch & Swofford, 2003) to reconstruct the replacement history of all polymorphic positions on the rooted phylogenetic trees. We used two maximum parsimony algorithms (ACCTRAN and DELTRAN) and considered only the events reconstructed by both algorithms.\n\nWe considered the first 18 amino acids of esxH (MSQIMYNYPAMLGHAGDM), which was by far the epitope with most amino acid replacements in L1. We expanded the analysis of this epitope to the L2 and L4 datasets, so that we could compare the results with L1 and L3. For the PAML analysis, we randomly selected 500 strains from each MTBC lineage. We used the phylogenetic tree reconstructed by RAxML (same settings as above), and the gene alignment to estimate the branch lengths of the tree using the M0 codon model implemented in PAML 4.9e (Yang, 2007). This step was necessary to obtain a tree with the branch length in expected substitutions per codon. We then fitted two alternative codon models (M1a and M2a) to the trees and alignments. M1a allows ω to be variable across sites, and it assumes two different ω (0 < ω0 < 1, and ω1 = 1), modeling nearly neutral evolution; M2a assumes one additional ω (ω2 > 1) compared to M1a, thus modeling positive selection. We performed a likelihood ratio test between the two models as described in Zhang et al. (2005). Templates for the control files of the codeml analyses (M0, M1a, M2a) are available as Supplementary Files in Extended data. The codon under positive selection were identified with the Bayes empirical Bayes method (Yang et al., 2005).\n\nTo test whether the derived haplotypes of this epitope were associated with a specific geographic region, we constructed a statistical test analogous to PhyC (Farhat et al., 2013), which is normally used to test for association between a variant and phenotypic drug resistance. We simulated mutations on the phylogenetic tree of L1 and counted how many strains from each region resulted to have a derived state according to the simulation. Under this procedure, mutations occur randomly on the tree, and therefore independently from the geographic region where the tips were sampled. For each test, we performed 10,000 simulations to obtain a null distribution, and we compared this distribution to the observed data to obtain empirical p-values. We used the same geographic region used for the biogeography analysis. Additionally, we considered East Africa excluding Madagascar.\n\nR code and input files to perform this test are available as Supplementary Files in Extended data.\n\nWe considered three HLA loci: HLA-A, HLA-B and HLA-DRB1. We used the allele frequency database (Gonzalez-Galarza et al., 2020) to identify alleles that are prevalent in East Africa and not in South Asia and Southeast Asia, or the other way around. Because the coverage of the allele frequency database is patchy, we focused on the following countries: Kenya and Zimbabwe as representatives of East Africa; India, Thailand and Taiwan as representatives of South- and Southeast Asia. We identified:\n\n1) Alleles that had a frequency of 10% or more in at least one population in South- and Southeast Asia, but had frequencies lower than 10% in all East African populations.\n\n2) Alleles that had a frequency of 10% or more in at least one population in East Africa, but had frequencies lower than 10% in all populations in South- and Southeast Asia.\n\n3) Alleles that had a frequency of 10% or more in at least one population both in South- and Southeast Asia and in East Africa.\n\nFor all these alleles, we performed in silico binding prediction with three epitopes: the ancestral epitope at the esxH N terminus (MSQIMYNYPAMLGHAGDM), and the two most frequently observed derived epitopes (MSQIMYNYPTMLGHAGDM, and MSQIMYNYPVMLGHAGDM). For HLA-A and HLA-B alleles, we used the NetMHCPan4.1 server (Reynisson et al., 2020) with standard settings. For HLA-DRB1 alleles, we used the prediction tool of the immune epitope database (https://www.iedb.org/).\n\nFor this analysis, we used the subsets generated for the Mascot analysis. These datasets are representative of the populations of L1 and L3 in their core geographic ranges, and are computationally treatable. We generated sequence alignments for all genes individually, excluding genes in repetitive regions of the genome (see above). Because some genes are deleted in L1 but not in L3, or the other way around, we obtained a slightly different number of gene alignments for the two lineages (L1: 3,623, L3: 3,622). For each gene, we performed a test for positive selection with PAML as described above for esxH. We considered as under positive selection all genes, for which the likelihood ratio test resulted in a Bonferroni-corrected p-value < 0.05.\n\nWe considered 196 mutations conferring resistance to different antibiotics (Payne et al., 2019). We extracted the respective genomic positions from the vcf file of the 4,968 genomes of the complete curated data set (2,938 L1, 2,030 L3) and assembled them in phylip format. To determine the number of independent mutations, we reconstructed the nucleotide changes on the phylogenetic tree rooted with the L2 strain (SAMEA4441446). To do this, we used the maximum parsimony ACCTRAN and DELTRAN algorithms implemented in PAUP 4.0a (Wilgenbusch & Swofford, 2003), and considered only the events reconstructed by both algorithms.\n\n\nData availability\n\nThe sequence data generated by this study has been deposited on SRA (https://www.ncbi.nlm.nih.gov/sra) under the accession numbers PRJNA630228 and PRJNA670836.\n\nExtended data is available here: https://github.com/fmenardo/MTBC_L1_L3.\n\nDOI: https://doi.org/10.5281/zenodo.4435760 (Menardo, 2021).\n\nThis project contains the following extended data:\n\nThe folder Supplementary_text_figures_tables contains supplementary text, figures and tables.\n\nSupplementary_file_1.html: PASTML results for L1, compressed tree.\n\nSupplementary_file_2.html: PASTML results for L1, complete tree.\n\nSupplementary_file_3.html: PASTML results for L3, compressed tree.\n\nSupplementary_file_4.html: PASTML results for L3, complete tree.\n\nThe folder Dating_Beast contains the xml files for the dating analyses.\n\nThe folder EsxH_haplotype_association_test contains the code and input files to perfomr the test of association between different haplotypes and geographic regions.\n\nThe folder EsxH_PAML contains the control files and input files to perform the positive selection analysis on EsxH.\n\nThe folder Mascot contains the xml files for the Mascot analyses.\n\nExtended data is available under a GNU GENERAL PUBLIC LICENSE.", "appendix": "Acknowledgements\n\nCalculations were performed at sciCORE (http://scicore.unibas.ch/) scientific computing core facility at the University of Basel.\n\n\nReferences\n\nAbel B, Tameris M, Mansoor N, et al.: The novel tuberculosis vaccine, AERAS-402, induces robust and polyfunctional CD4+ and CD8+ T cells in adults. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "78953", "date": "17 Feb 2021", "name": "Tanvi Honap", "expertise": [ "Reviewer Expertise Pathogen genomics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe aim of this study was to conduct a phylogeographic analysis of Mycobacterium tuberculosis complex (MTBC) strains belonging to Lineages 1 and 3, which are the most prevalent lineages in South Asian countries with a high TB burden, such as India. The authors compiled an excellent dataset of nearly 5,000 MTBC genomes from these two lineages, comprising publicly available as well as newly-sequenced MTBC strains. Their phylogenomic analyses showed that L1 comprises a further five distinct sublineages, whereas L3 does not contain distinct sublineages. The authors used two different methods (PASTML and Mascot) for conducting phylogeographic analyses, with results from both methods suggesting an interesting asymmetric pattern of migration: these lineages originated in and migrated from South Asia to other regions, with very little back-migration into South Asia. This underscores the important role South Asia has played in the dispersal of these lineages. The authors found several genes involved in virulence and antibiotic resistance to be under positive selection in L1 and L3 strains. Lastly, the authors also found evidence of local adaptation at the esxH epitope in L1 strains from South and Southeast Asia, which requires further study as this epitope is a part of several vaccine candidates.\n\nOverall, I found this to be a well-designed and thorough study, and a very well-written manuscript. I have no major concerns and only a few minor suggestions for aesthetic improvement:\nThe Methods section titled \"Strain cultures, DNA extraction and genome sequencing\" is missing citations for the methods used (eg. CTAB extraction method).\n\nFigures 1A and 2A heatmaps would look better in color than grayscale.\n\nThe Supplementary Information would benefit from a thorough proofreading for grammatical and typological errors.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6476", "date": "29 Mar 2021", "name": "Fabrizio Menardo", "role": "Author Response", "response": "Thank you very much for your comments, and for reviewing our manuscript. We prepared a revised version of the manuscript based on the feedback of all reviewers. We added the missing reference for the DNA extraction method, and we revised the supplementary material for errors and typos, thanks. Regarding Figures 1A and 2A: in both figures the other three panels have the same color code, where different colors correspond to different world regions. We decided to use a gray scale to avoid potential confusion of having two different color codes in the same figure. We did not modify the figures." } ] }, { "id": "79237", "date": "01 Mar 2021", "name": "Miguel Viveiros", "expertise": [ "Reviewer Expertise Mycobacteriology", "Tuberculosis", "Laboratory Diagnosis and Molecular Epidemiology." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study depicts a very interesting and relevant account on the evolutionary history and phylogeography of Mycobacterium tuberculosis of Lineages 1 and 3. Historically, and while forming an important part of the M. tuberculosis species diversity, the study of both lineages have been neglected in comparison to Lineages 2 and 4. The authors of this study assembled a large genomic dataset encompassing 2938 and 2030 L1 and L3 isolates, respectively, and by implementing distinct molecular evolutionary algorithms were able to put forward a scenario for the dissemination of these strains. A South Asian origin is proposed for both clades from where these strains appeared to have radiated via migration-driven dispersion towards East Africa (L3) and other regions (L1). The weakest aspect of the work is the lack of phylogeographic and historical context of L1 and L3 versus L2 and L4 dissemination, especially L4 in South-America and L2 in Asia in the context of the migratory waves.\nThe authors used PASTML and Mascot to conduct the phylogeographic analyses, revealing the migratory pattern of these lineages from South Asia revealing the origins of these two lineages. Several genes involved in virulence and drug resistance were shown to be under positive selection in L1 and L3 strains with important adaptative consequences. Importantly, esxH derived haplotypes were not randomly distributed through the geographic range of L1 and the finding of positive selection acting on vaccine candidates, such as this, is important and deserves attention.\nThe manuscript is very well written, easy to read and understand and the methods are robust and well applied and described. Important to note the absence of a few important references to other groups work on the third wave migration of migrations in the 16th century that impacted the dissemination of L1 in Africa and other regions.\nSome comments that can be useful for discussion:\nThe analysis pertaining the introduction of L1 to South America is based on a more modest number of isolates (n=77), all but one from Brazil. This limits the robusteness of the conclusions for this region. It is possible that the evolutionary history of L1 in South America is far more complex than the one outlined in the article and there may be missing links. For example, EAI (L1) strains have been detected in several South and Central American countries (Mexico, Peru, Panama, Colombia, etc.) and besides slave trade, the Spain-operated Manila-Acapulco Galleon trade route (est. ca. 1568) connected Southeast Asia to Central and South America.\nRegarding the L1.1.1 sub-lineage, what precludes these strains to have been introduced to West Africa in a more recent period other than the XVI or the XVII century? As far as we can tell, this pertains a group of 10 isolates scattered over five countries, but what is the degree of divergence between these isolates? Eventually, can this also be linked with the indentured labor promoted by the British Empire in the XIX century?\nOn the epitope analysis, upon downloading epitopes from IEDB did the authors include only epitopes with positive assays and was there a threshold for including only epitopes validated by independent assays (more reliable)? Also, how did overlapping epitopes been handled?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6477", "date": "29 Mar 2021", "name": "Fabrizio Menardo", "role": "Author Response", "response": "Thank you very much for reviewing and commenting our manuscript. We prepared a revised version of the manuscript based on the feedback of all reviewers. Regarding the comparison with other lineages: in this manuscript we focused on L1 and L3 because they are understudied. We now revised the text and added a section comparing our findings with the biogeography of other MTBC lineages, and referencing the relevant studies. Additionally, it is true that the evolutionary history of L1 in South America could be more complex, and we might fail to capture it because we have samples from a single country (Brazil). We now explicitly write this. Regarding the L1.1.1 sub-lineage: in the manuscript we write that the 16th century is the earliest possible time for the introduction of L1 in West Africa (assuming that the migration could not have happened before the circumnavigation of Africa by Portuguese), we do not exclude the possibility of a later introduction. However, this group of 11 L1 strains comprising the West African clade has a relatively old MRCA. To be compatible with an introduction in the 19th century, the molecular clock rate would need to be about 3x10-7 (this is a back of the envelope calculation, and not the result of a proper analysis). This would be a fast rate for MTB (Menardo et al. 2019), and we find this hypothesis unlikely. Nevertheless, because of the lack of a temporal signal we cannot exclude a more recent introduction of L1 in West Africa. Finally, in the epitope analysis we downloaded all T cell epitopes, to be as inclusive as possible. Overlapping epitopes were analyzed independently. We added this information to the manuscript." } ] }, { "id": "79710", "date": "15 Mar 2021", "name": "Ola B. Brynildsrud", "expertise": [ "Reviewer Expertise Bioinformatics", "microbial genomics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the paper \"Local adaptation in populations of Mycobacterium tuberculosis endemic to the Indian Ocean Rim\", Menardo and colleagues explore diversity, phylogeographic relationships and patterns of adaptation in TB lineages 1 and 3, both of which are distributed mainly around the Indian Ocean. The paper demonstrates a strongly asymmetric trend of South Asian spread to other regions. Further, it describes a search for positive selection throughout these genomes, and identify one gene in particular, exsH, as being a target for T-cell dependent selection. The authors additionally calibrates molecular clocks for both L1 and L3 and use this to date certain events in the tree.\nThe methodology is solid and well suited to answer the questions posed. I found the paper to be well written and helpful in shedding light on important questions related to the evolution of L1 and L3. I have only a few minor discussion points.\nNow, the clock analyses are not heavily emphasized, but I thought it might be a potential flaw that the calibration rests on the assumption that the foundation of a single sub-lineage in West Africa could not have happened prior to the establishment of Portuguese sailing routes around the tip of Africa. While I'm certainly not an expert on historic migrations or anthropology, I would assume there must be many possible alternative scenarios. (For example, pilgrimages across the Sahara are known to have happened much prior to this). As far as I can tell, this calibration is the major driving force for the difference in L1 TMRCA from O'Neill. It would be interesting to know how the degree to which this calibration affects the TMRCA and clock rate. On a somewhat related note, this article fails to cite some key references on the phylogeography of TB.\nIn one section the authors evaluate association between esxH haplotypes and the geographic region of origin. They calculate this association using a home-made statistical test based on phyC. I could not gather from the description exactly what they measure. It is a simulation-based test that measures \"how many strains from each region resulted to have a derived state according to the simulation\". Is there a test statistic being ranked here? Is it single-region vs the rest or is the statistic a composite measure of multiple regions? How is the simulation for the null distribution set up? E.g. is it a forward-in-time simulation using a specified mutation rate or a permutation or something else? I see that the code for running this test is posted on github - However, I still think a few more details in the actual paper is in order for the biologists that don't read R. Speaking of this test, It is a bit unconvential for Table 1 to highlight all results with P<0.1, especially since a large number of associations are being tested here. I would not consider p<0.1 to be particularly strong evidence of association.\nI'd like to commend the authors for making so much of their data publicly available. Datasets are in immediately-usable formats which makes it simple to download all the raw data, and as mentioned code is also readily available.\n\nVery minor:\nHLA abbreviation never explained in manuscript.\n\nTip date randomisation performed, but the results of this test is never stated.\n\nWhy is PRJNA630228 posted? As far as I can tell it doesnt contain any SRA data, and the other accession contains data for all 767 strains.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6478", "date": "29 Mar 2021", "name": "Fabrizio Menardo", "role": "Author Response", "response": "Thank you very much for your comments, and for reviewing our manuscript. We address all your points below. We prepared a revised version of the manuscript based on the feedback of all reviewers. Regarding the clock analysis of L1: We performed a tip dating analysis but found no temporal signal for L1. Therefore we used the clock rate estimate resulted from a previous study (Menardo et al. 2019) where we analyzed a different L1 dataset and found a good temporal signal and a clock rate of ~1.4x10-7. So far Menardo et al. 2019 is the only published study that estimated the clock rate for a L1 dataset. In a second step we tested whether this clock rate and two alternative values, 5x10-8 and 1.2x10-7,, (the lowest one was used by O’Neill et al. 2019) were compatible with the hypothesis of a West African introduction of L1.1.1 posterior to the Portuguese circumnavigation of Africa. We found that only the largest clock rate was compatible with this hypothesis. We are quite explicit in the manuscript about the limitation of this analysis, and how it should be interpreted in comparison to the results of O’Neill et al. 2019: “This discrepancy is due to different assumptions about the clock rates of L1, but none of the two hypotheses can be excluded with the available data”. Finally, it is possible that the West African L1.1.1 clade did not originate through a direct migration from South East Asia. However, a series of short range migrations seems less likely, as we did not find strains belonging to L1.1.1 in South and Central Asia, or in East Africa. As we write in the manuscript, the dating analyses for MTB in general, and for this L1 dataset in particular, have limitations. Here we discussed what we consider the most likely scenario given the data, we did not exclude alternative scenarios. We added a section in the discussion comparing our findings with the biogeography of other MTBC lineages, and referencing the relevant studies. We revised and expanded the Methods section regarding the phylogenetic test of association to provide more details. To answer the specific questions: the statistic ranked is the number of tips with a derived state; the test is performed for each region individually; the simulation does not use a mutation rate, but it distributes randomly a fixed number of mutations on the tree (the same number inferred from the observed data). We also removed special fonts from Table 1, now all p-values have standard formatting. Minor points: We now write HLA in full at the first occurrence. The results of the DRT are reported in the supplementary Information (now amended to enhance clarity), in Sup. Figs 8 and 9 and in Sup. Table 3. In the main text we refer more generally to the lack of temporal signal (or to a good temporal signal), as this reflects the results of the DRT, but also of the Beast analyses. We now refer only to PRJNA670836, which contains data for all the newly sequenced strains. Thanks." } ] } ]	1	https://f1000research.com/articles/10-60
https://f1000research.com/articles/10-113/v1	15 Feb 21	{ "type": "Research Article", "title": "The clinical impact of bacterial co-infection among moderate, severe and critically ill COVID-19 patients in the second referral hospital in Surabaya", "authors": [ "Tri Pudy Asmarawati", "Alfian Nur Rosyid", "Satriyo Dwi Suryantoro", "Bagus Aulia Mahdi", "Choirina Windradi", "Prastuti Asta Wulaningrum", "Muhammad Vitanata Arifijanto", "Bramantono Bramantono", "Erwin Astha Triyono", "Musofa Rusli", "Brian Eka Rachman", "Erika Marfiani", "Pepy Dwi Endraswari", "Usman Hadi", "Kuntaman Kuntaman", "Nasronudin Nasronudin", "Satriyo Dwi Suryantoro", "Bagus Aulia Mahdi", "Choirina Windradi", "Prastuti Asta Wulaningrum", "Muhammad Vitanata Arifijanto", "Bramantono Bramantono", "Erwin Astha Triyono", "Musofa Rusli", "Brian Eka Rachman", "Erika Marfiani", "Pepy Dwi Endraswari", "Usman Hadi", "Kuntaman Kuntaman", "Nasronudin Nasronudin" ], "abstract": "Background: Data on the prevalence of bacterial co-infections among COVID-19 patients are limited, especially in our country, Indonesia. We aimed to assess the rate of bacterial co-infections in hospitalized COVID-19 patients and report the most common microorganisms involved and the antibiotic use in these patients. Methods: This study is a cross sectional study with retrospective approach, among COVID-19 adult patients admitted to Universitas Airlangga Hospital Surabaya from 14 March-30 September 2020. The bacterial infection is defined based on clinical assessment, laboratory parameters, and microbiology results. Results: A total of 218 patients with moderate to critical illness and confirmed COVID-19 were included in this study. Bacterial infection was confirmed in 43 patients (19.7%). COVID-19 patients with bacterial infections had longer hospital length of stay (17.6 ± 6.62 vs 13.31±7.12), a higher proportion of respiratory failure, intensive care treatment, and ventilator use. COVID-19 patients with bacterial infection had a worse prognosis than those without bacterial infection (p<0.04). The empirical antibiotic was given to 75.2% of the patients. Gram-negative bacteria were commonly found as causative agents in this study (n = 39; 70.37%). Conclusion: COVID-19 patients with bacterial infection have a longer length of stay and worse outcomes. Healthcare-associated infections during intensive care treatment for COVID-19 patients must be carefully prevented.", "keywords": [ "Bacterial infection", "COVID-19", "SARS-CoV-2", "antibiotics" ], "content": "Introduction\n\nCoronavirus disease (COVID)-19 has experienced an increase in 2,995,758 positive cases and 204,987 deaths in distribution areas of more than 213 countries1. In Indonesia, until November 2020, there were 522,581 confirmed cases of COVID-19 with 68,604 active cases, 437,456 recovered cases, and 16,521 (3%) deaths. According to national data, among the total number of cases, East Java is the second-highest prevalence of 60,190 (11.6%). As of November 26, 2020, the cumulative data on confirmed COVID-19 patients in Surabaya were 16,763 with 1204 (7.03%) deaths1.\n\nData regarding secondary respiratory infection in COVID-19 patients are still limited even though cases distribution is still worldwide. Several reports suggest that secondary infection will impact the patient’s survival and increase intensive care unit (ICU) treatment2. COVID-19 pneumonia itself is also related to increasing ICU care, secondary infection rate, and higher invasive treatment3. Co-infection of SARS-CoV-2 and other microorganisms such as viruses, bacteria, and fungi is an essential factor in COVID-19 treatment since this condition may raise the difficulty in diagnosis, treatment, and the prognosis, also increasing the mortality4,5. Upper respiratory tract infection, a typical manifestation of COVID-19, is challenging to differentiate from other causes of pneumonia.\n\nThere are various types of co-infection in COVID-19; such as: 1) secondary SARS-CoV-2 following bacterial infection or colonization; 2) mixed infection between viral and bacterial pneumonia infection; 3) secondary bacterial superinfection following SARS-CoV-2 infection. The mechanisms underlying those types of infection are very dependent on the onset and involving complex interactions between three different agents (virus, host, and bacteria). Immune response towards SARS-CoV-2 infection only differs with mixed infection to bacteria or viral pneumonia. Therefore, it could be hypothesized that any co-infection will worsen the outcome and severity of COVID-196.\n\nAlthough several studies have investigated the epidemiological and clinical characteristic of COVID-19, information regarding SARS-CoV-2 infection with secondary infection are limited7. This study aims to describe bacterial co-infection and antibiotic use among patients who confirmed SARS-CoV-2 infection from moderate to critically ill manifestation in Universitas Airlangga hospital Surabaya.\n\n\nMethods\n\nThis study is a retrospective cohort study, total sampling, among COVID-19 adult patients admitted to Universitas Airlangga Hospital Surabaya. This hospital is an academic hospital and also a referral hospital for COVID-19 management in Surabaya, East Java Region. We included cases of moderate to critically ill COVID-19 patients between 14 March and 30 September 2020 that were admitted in the intensive care unit or high-care unit. COVID-19 diagnosis was made based on World Health Organization (WHO) guidelines8 and the Indonesian Ministry of Health guidelines9. Confirmed COVID-19 patients were proven by oropharyngeal and nasopharyngeal swabs SARS-CoV-2 PCR (polymerase chain reaction). Bacterial infection was defined based on clinical assessment, laboratory parameters, and inflammatory parameters (C-reactive protein (CRP) and procalcitonin). Bacterial co-infection of SARS-CoV-2 defines if the culture samples were taken at patient presentation to the hospital or < 48 hours admission, while secondary bacterial infection defined if the culture samples were taken > 48 hours of admission. The bacterial causative agents were extracted from data of microbiology, that were identified by Microbiology automated machine Vitek-2 compact, as a routine procedure in this hospital.\n\nThis study was approved by the Ethical Committee of Universitas Airlangga Hospital (171/KEP/2020). Written informed consent was obtained from all participants prior to the start of the study.\n\nData were taken from medical records and microbiology reports from the laboratory. Incomplete medical records were excluded. Clinical characteristics were divided according to the severity of the disease. Moderate case definitions are: 1) clinically sign of pneumonia (fever, cough, dyspnea, tachypnea); 2) Oxygen saturation ≥93% free air. Severe case definitions are if there were clinically sign of pneumonia, and one of the following: 1) respiration rate >30 times per minute, or 2) severe respiratory distress, or 3) oxygen saturation < 93% free air. Critically ill cases defined if there were acute respiratory distress syndrome (ARDS), sepsis, and septic shock10. Culture examination was performed when there was suspicion of bacterial infection or sepsis. The sample for culture was taken from blood, urine, and respiratory tract.\n\nData were analyzed with SPSS version 24.0 (Chicago, IL, USA). Descriptive statistics included categorical variables reported as number (percentage) and continuous variables as mean (standard deviation). For missing data, we used listwise deletion or univariable and multivariable analysis. Chi-square test and Mann-Whitney test were used accordingly to the type of variable. Categorical variables were shown as number (%) and continuous variables as mean (standard deviation) or median (range) depending on whether the data are normally distributed or not. Statistical significance was assessed by means of chi-squared for dichotomous variables, or by means of the two independent sample t-test or the Mann-Whitney U test for continuous variable depending on whether the data are normally distributed or not.\n\n\nResults\n\nFrom March 14 until September 30, 2020, a total of 218 patients confirmed for SARS-CoV-2 infection were admitted to Universitas Airlangga Hospital Surabaya from moderate to critically ill condition. Patients characteristic symptoms were defined according to their severity. Clinical characteristics and main comorbidities are detailed in Table 1. The median age of the study subject was 52.45 (±14.44) years, and 55.05% of patients were male. According to disease severity, the number of patients with moderate, severe, and critically ill manifestations were 126 (57.8%), 40 (18.3%), and 52 (23.9%), respectively. Diabetes and hypertension were the most common comorbidity, in 34.4% and 29.9% of patients, respectively. Among all subjects, patients that were critically ill 7.3% manifested respiratory failure (p <0.05), 23% were on a ventilator (p <0.05) and in 7.8% were in sepsis (p = 0.006). Critically ill patients had the longest length of stay (mean 16.89 ± 9.4 days). Bacterial infection was confirmed in 43 patients (19.7%); 16 patients were in critically ill condition. Among COVID-19 patients with a bacterial infection, we divided them into two categories, bacterial co-infection (23%) and secondary bacterial infection (77%).\n\nCharacteristic symptoms and laboratory result are shown in Table 1. The most common symptoms in all severity were dyspnea, fever, cough, and malaise. In patients who were moderate, severe, and critically ill, symptoms cough (7.34%, 3.21%, 0.92%), fever (16.97%, 3.21%, 2.75%) and dyspnea (19.27%, 7.34%, 14.68%) respectively. Other symptoms recorded were nausea, vomiting, diarrhea, headache, joint pain, chest pain, and loss of consciousness, but only affected the minority of the patients. From vital sign examination, two variables had a significant difference for moderate, severe, and critically ill patients; respiratory rate (23.34, 24.73, 28.92; p <0.05) and oxygen saturation (SaO2) (94.2, 88.05, 88.24; p <0.05) respectively. Some symptoms vary in duration between groups. Severe patients complained of cough symptoms for an average of 10.6 days, longer than those in the moderate or critically ill group. The duration of fever was almost similar between groups, namely 6–8.8 days.\n\nFrom the laboratory findings, the mean leukocyte count (p = 0.014), neutrophil count (p = 0.001), procalcitonin (p = 0.033), basal urea nitrogen (BUN) (p = 0.007), alanine aminotransferase (ALT) levels (p = 0.01) were significantly higher in either severe or critically ill COVID-19 patients than moderate cases. We also found a lower lymphocyte count (p = 0.001) and PaO2: FiO2 ratio (p = 0.005) in either severe or critically ill COVID-19 patients than moderate cases. The procalcitonin level significantly increased in severe and critical illness conditions. The CRP level also increased but was not statistically significant. The majority of patients (82.9%) were recovered and discharged from the hospital, while 9.6% of the patient died. Most of the patients who died were in critically ill condition at presentation to hospital.\n\nBacterial infection was confirmed in 43 patients (19.7%) (see Table 2). There were no sex differences between bacterial infection and no bacterial infection patients. Patients with a bacterial infection have an older mean age than no bacterial infection, although, among elderly patients, there were no differences in bacterial infection rate. We documented one patient with chronic obstructive pulmonary disease (COPD) and one with liver disease as a comorbidity, and both of them suffered from bacterial infection. Other comorbidities such as diabetes, hypertension, heart disease, stroke, chronic kidney disease did not differ between the two categories. COVID-19 patients with bacterial infections had a longer hospital length of stay, a higher proportion of respiratory failure, ICU treatment, ventilator use.\n\nMortality occurs in 16.28% of COVID-19 patients with bacterial infections, higher than those without bacterial infection (8%). Overall, COVID-19 patients with bacterial infection had a worse prognosis than those without bacterial infection (p<0.04).\n\nThe empirical antibiotic was given to 75.2% of the patients. Antibiotics used in these patients were quinolones (60.1%), cephalosporins (28.44%), carbapenem (23.85%), and aminoglycosides (4.59%). Quinolone used in these patients was mostly levofloxacin (79.39%), and the others were moxifloxacin (20.61%). The carbapenem used in this study was meropenem. Cephalosporins used were ceftriaxone, ceftazidime, cefotaxime, cefoperazone-sulbactam, and cefuroxime (see Figure 1).\n\nWe collected 110 culture samples from suspected bacterial infection patients, consisting of 44 blood samples, nine urine samples, and 57 sputum samples. Among them, bacteria were detected in nine blood cultures, four urine cultures, and 47 sputum cultures. Sputum samples were collected, some from spontaneous sputum (n=47) and the others from endotracheal tube aspirate (n=10). Gram-negative bacteria dominate the culture result (70.37%). Bacteria found in the blood were extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae (2), Pseudomonas fluorescens (1), Pseudomonas putida (1), Staphylococcus epidermidis (2), Staphylococcus haemolyticus (1), Staphylococcus hominis (2). Bacteria found in urine were ESBL-producing Escherichia coli (2), Enterococcus faecalis (1), and Pseudomonas putida (1). There were 47 isolates found in sputum cultures, in which six of them were fungi (Candida spp). The most frequent bacteria found was Acinetobacter baumannii (9), followed by Klebsiella pneumoniae (5), Pseudomonas aeruginosa (4), Escherichia Coli (2), Enterobacter cloacae complex (3), and Staphylococcus haemolyticus (3) (see Table 3 and Figure 2).\n\nNote : *, considered as normal flora bacteria.\n\n\nDiscussion\n\nOur study focuses on bacterial infection results in COVID-19 patients and evaluates their clinical and microbiological features. Clinical characteristics of the study group were described according to disease severity. According to disease severity, the number of patients with moderate, severe, and critically ill manifestations were 126 (57.8%), 40 (18.3%), and 52 (23.9%), respectively. The average age was 52.45 (±14.44) years, and 55.05% of patients were male. Other studies regarding the characteristic of hospitalized COVID-19 patients showed various results. Zhou et al. reported that the median age of the 191 patients was 56.0 years (18–87 years), and most patients were male11. Lv et al. reported 354 hospitalized patients, 175 (49.44%) were male, and the median age was 62 years (23–90 years)12. A study conducted in Saudi Arabia reported among the 99 hospitalized patients, the median age was 44 years (range 19–87), and the majority were men (66%)13. We found that diabetes and hypertension were the most common comorbidity, similar to these other studies11–14.\n\nIn this study, inflammation signs such as leukocyte count, neutrophil count, procalcitonin in the critically ill group were higher than the others. Lymphopenia in COVID-19 patients occurs through various mechanisms such as direct virus invasion lymphocytes, lymphatic organ destruction, altered inflammatory cytokines production leading to lymphocyte apoptosis, and inhibiting lymphocytes function by metabolic molecules produced by metabolic disorders, such as hyperlactic academia15. Neutrophil and lymphocyte ratio (NLR) has been proposed as a prognostic marker of severity in various chronic inflammatory diseases, including cardiovascular diseases and oncological processes16.\n\nOur result showed a significant increase of procalcitonin level and a higher proportion of bacterial infection in severe and critically ill COVID-19 patients. This finding is consistent with the study result from Wang et al. in elderly COVID-19 patients. They also concluded that bacterial infection was also considered as a predictor of mortality in these patients17. Increased procalcitonin level in COVID-19 patients may be associated with the release of some cytokines, especially IL-6. It is established that procalcitonin is a better marker to predict severity, prognosis, or the sepsis course and is also helpful to guide antibiotic usage. Increased procalcitonin in critically ill patients with COVID-19 can represent a bacterial co-infection, and blood cultures for bacteria detection are needed to a prompt response18.\n\nIn this study, we found that bacterial infection was confirmed in 43 patients (19,7%). The bacterial infection that was detected at patient admission (bacterial co-infection) was 23%, while bacterial infection that occurs late during hospital stay (secondary infection) was 77%. The prevalence of bacterial co-infections in patients admitted to the ICU for acute respiratory failure related to COVID-19 pneumonia is poorly studied19,20. In Fu et al. study, 13.9% (5 of 36) of the patients in the ICU were diagnosed with COVID-19 and secondary bacterial infection. In another report that was published from a UK secondary care setting, 27 among 836 patients (3.2%) had early confirmed bacterial isolates identified (0–5 days post-admission), rising to 51 cases (6.1%) during the admission. In a study conducted in Shiraz, Iran, in 2009, Hassanzadeh et al. suggested that ICU-acquired infections were documented in 51.7% of ICU patients, with a mortality rate of 10.9% (5 patients)21,22. Our finding was relatively higher among other studies. The possible explanation is that most of the patients had been treated in hospital for more than two weeks (average 12–17 days). Secondary infections usually correspond with nosocomial or healthcare-associated infections. Nosocomial infections are most commonly correlated with invasive medical devices or surgical procedures. Lower respiratory tract and bloodstream infections have the highest mortality, while urinary tract infections are the most common23.\n\nThe median length of stay among patients in our study was higher in the bacterial co-infection group rather than non co-infection, with an average of 17.6 and 13.31 days, respectively. This finding is consistent with the result of several studies21,24. Sharifpour et al. reported that the median length of stay is around 15 days (interquartile range, 2 to 39). A study on respiratory co-infection in patients with pandemic 2009 influenza A (H1N1) virus infection showed that ICU length of stay was three days longer among patients with co-infection. Another study by Zhou et al. reported a longer length of stay of 8.0 days (4.0–12.0) of all patients with COVID-19 admitted to their ICU11. These findings suggest that the length of ICU stay can be prolonged if patients become co-infected.\n\nThe most common bacterial infection in this study was from respiratory tract infection, followed by bloodstream infection and urinary tract infection. The culture result was dominated by gram-negative bacteria (75.68%). This finding is similar to the result from Zhang et al. (50%), although they also included virus and fungal cultures3. Gram-negative bacteria were reported responsible for more than 30% of healthcare-associated infections, and these bacteria predominate in cases of ventilator-associated pneumonia (47%) and urinary tract infections (45%)23. In intensive care units (ICUs), gram-negative bacteria account for about 70% of these types of infections25. Gram-negative bacteria are highly efficient at up-regulating or acquiring genes that code for antimicrobial resistance mechanisms, especially in the presence of antibiotic selection pressure. Among gram-negative bacteria, the Enterobacteriaceae family being the most commonly found and multidrug-resistant organisms, including Pseudomonas aeruginosa, Acinetobacter baumannii, and extended-spectrum β- lactamase (ESBL)–producing or carbapenemase-producing Enterobacteriaceae, are increasingly being reported worldwide23. The identification of bacterial infection with gram-negative organisms is more reflected as a complication of ICU care and is not suggested as a specific predilection for co-infections in COVID-1922.\n\nAcinetobacter baumannii is the most common bacteria found in this study. Several reports regarding bacterial infection in COVID-19 also found a similar result3,7,9,18,23. Acinetobacter baumannii is the common cause of respiratory tract infection, especially in ICU settings where patients often received mechanical ventilation. Environmental contamination also plays a role in this phenomenon. Our ICUs for COVID-19 patients were set as a large ward consist of 16 beds. Although we already managed the distance between beds for more than 1.5 meters, the inter-patient transmission is hard to avoid. It is crucial to maintain hand hygiene compliance of the staff, strict cleanliness, and disinfection of the hospital environment in the hospital, especially in high-care or ICU setting.\n\nAntibiotic is often given to COVID-19 patients for various reasons. The clinical manifestation on presentation was sometimes challenging to distinguish with bacterial infection2. Secondary infection and nosocomial infection are also other considerations in the use of antibiotics. In this study, 75.3% of patients included were given antibiotics. A meta-analysis conducted about bacterial co-infection and secondary infection in COVID-19 patients reported that the majority of patients with COVID-19 received antibiotics (71.9%, 95%CI 56.1 to 87.7%)26. Another meta-analysis reported that >90% of COVID-19 patients were given an empirical antibiotic, while the bacterial infection was detected only in 7% of hospitalized patients and 14% of ICU patients27. Quinolones (levofloxacin and moxifloxacin) were frequently prescribed, followed by cephalosporins and carbapenems. Indonesian national guidelines for COVID-19 treatment recommend using antibiotics in severe and critically ill patients, especially if there was suspicion of bacterial infection. The empirical antibiotic choice was intravenous azithromycin or levofloxacin10. In our hospital, levofloxacin is more feasible than azithromycin, so clinicians prefer to prescribe this drug. In our hospital, antibiotic use usually was decided based on clinical signs and laboratory parameters (leukocytosis, increased CRP, or procalcitonin). Culture examinations were not routinely performed, preferably in ICU/high-care settings and in patients showing bacterial infection.\n\nThe widespread use of antibiotics in this pandemic era raises concern about antimicrobial resistance4,6. Although bacterial infection in COVID-19 patients has already been reported in various results, evidence supports the restrictive use of antibiotics. Clinical guidelines and standard testing to diagnose bacterial infection in COVID-19 are not clearly available. The microbiological examination is an important strategy to confirm bacterial infection and decide the antibiotic choice4. Sputum, blood culture samples, and also pneumococcal urinary antigen testing should also be performed. Antibiotics should be stopped in patients who represent cultures, and urinary antigen tests show no signs of bacterial pathogens after 48 hours28.\n\nThere are some limitations to our study. First, we only included COVID-19 patients in high-care and intensive care units. In low-care settings, bacterial infection cannot be confirmed due to limited data. Second, we only reported bacterial infection from microbiology results. Virus culture or gene sequencing to detect pathogens is not available in our hospital. Bacterial culture was not performed in all COVID-19 patients, so in a condition where the sign of infection is absent such as in elderly or in immunocompromised patients, the bacterial infection might be underdiagnosed.\n\n\nConclusions\n\nBacterial infection was detected in 19.7% of COVID-19 patients admitted in high-care and intensive care units, predominantly secondary infections. COVID-19 patients who suffered from bacterial infection have a longer length of stay and have higher mortality. The pathogen commonly found in this study was Acinetobacter baumannii that yielded from sputum. Increased antibiotic use and multi-drug resistant organism is an emerging problem in this pandemic situation. Infection control practice need to strictly conducted to reduced secondary or healthcare-associated infection\n\n\nData availability\n\nDryad: The clinical impact of bacterial co-infection among moderate, severe and critically ill COVID-19 patients in the second referral hospital in Surabaya, https://doi.org/10.5061/dryad.sxksn032829.\n\nThis project contains the following underlying data:\n\n- Baseline characteristics for all patients\n\n- Lab data for all participants\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgments\n\nWe would like to thank Esthiningrum Dewi Agustin, Jamiatul Khairiyah, Nila Novia Putri, Adhyasta Nata Prawira S for supporting this research.\n\n\nReferences\n\nSatuan Tugas Penanganan COVID-19: Peta Sebaran COVID-19 Satgas Penanganan COVID-19. 2020.\n\nFattorini L, Creti R, Palma C, et al.: Bacterial coinfections in COVID-19: an underestimated adversary. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSharifipour E, Shams S, Esmkhani M, et al.: Evaluation of bacterial co-infections of the respiratory tract in COVID-19 patients admitted to ICU. BMC Infect Dis. 2020; 20(1): 646. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLansbury L, Lim B, Baskaran V, et al.: Co-Infections in People with COVID-19: A Systematic Review and Meta-Analysis. SSRN Electron J. 2020. Publisher Full Text\n\nPeleg AY, Hooper DC: Hospital-Acquired Infections Due to Gram-Negative Bacteria. N Engl J Med. 2010; 362(19): 1804–13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRothe K, Feihl S, Schneider J, et al.: Rates of bacterial co-infections and antimicrobial use in COVID-19 patients: a retrospective cohort study in light of antibiotic stewardship. Eur J Clin Microbiol Infect Dis. 2020; 1–11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGaynes R, Edwards JR: Overview of Nosocomial Infections Caused by Gram-Negative Bacilli. Clin Infect Dis. 2005; 41(6): 848–54. PubMed Abstract \| Publisher Full Text\n\nLangford BJ, So M, Raybardhan S, et al.: Bacterial co-infection and secondary infection in patients with COVID-19: a living rapid review and meta-analysis. Clin Microbiol Infect. 2020; 26(12): 1622–1629. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLansbury L, Lim B, Baskaran V, et al.: Co-infections in people with COVID-19: a systematic review and meta-analysis. J Infect. 2020; 81(2): 266–75. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSieswerda E, de Boer MGJ, Bonten MMJ, et al.: Recommendations for antibacterial therapy in adults with COVID-19 - an evidence based guideline. Clin Microbiol Infect. 2021; 27(1): 61–66. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAsmarawati TP, et al.: The clinical impact of bacterial co-infection among moderate, severe and critically ill COVID-19 patients in the second referral hospital in Surabaya, Dryad. 2021. http://www.doi.org/10.5061/dryad.sxksn0328" }	[ { "id": "80840", "date": "09 Mar 2021", "name": "Fitriana Murriya Ekawati", "expertise": [ "Reviewer Expertise Health service research in LMICs" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you very much for the opportunity for reviewing this manuscript. This manuscript has presented interesting findings of bacterial coinfection in COVID-19 patients. Data collected for the research were from patients' medical records in Univ Airlangga hospital in Indonesia. The presentations of Methods, Results, and Discussion in the manuscript are appropriate. However, there are some minor revisions needed, which I believe are beneficial for improving the manuscript:\nIntroduction\n- Line 1. I would recommend the authors to improve the first sentence of the first and second paragraph to improve clarity.\n\n- Are there any prior investigations related to this topic in Indonesia? Please write/present if in the introduction there is any.\nMethods\n- Just to clarify, so the infection was examined if there is any suspicion of bacterial infection and not a routine examination?\n- Statistical analysis: SPSS version 24.0 (citation?).\nResults\n- Patient characteristic and laboratory findings, is there any table/summary table associated with the patients' leucocyte count, procalcitonin? Please cite/involve the summary table in the main manuscript.\n\nDiscussion\n- Are there any things that still unknown/need further investigation arising from this research?\n- What are the study implications for further research and practice?\n- I would recommend a language proofreading for this manuscript.\nBest wishes to the authors for the manuscript revision.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6426", "date": "29 Mar 2021", "name": "Tri Pudy Asmarawati", "role": "Author Response", "response": "We firstly would like to thank you for the kindly comprehensive review of our manuscript. Here's to answer and respond to your questions: 1. Introduction We believe that there is no prior investigation linked to this topic ever done before in Indonesia 2. Methods We clarify that the bacterial culture examination was done on the suspicious patients only. 3. Results We have enclosed the leucocyte and procalcitonin data of the patients on the table 1. 4. Discussion In the study to come, further investigation can probably be done on the low health-care setting, also bigger concerns on under-diagnosed bacterial co-infection of elderly and immunocompromised patients can be taken into consideration. The implication of the study is that we find the bacterial culture examination necessary in the COVID-19 patients management since the bacterial co-infection might worsen the clinical outcome. Furthermore, we also appreciate your suggestions in order to improve the final quality of this manuscript. We are currently working on the minor revisions, proof-reading, and going to submit the latest version promptly." }, { "c_id": "6493", "date": "29 Mar 2021", "name": "Tri Pudy Asmarawati", "role": "Author Response", "response": "We have just process revised several points on the manuscript as what the reviewer, named Fitriana Murriya Ekawati, had recommended a couple days ago. We include the latest version of our manuscript down below along with highlighted changes. We also insert our reply to the reviewer, which said: “We firstly would like to thank you for the kindly comprehensive review of our manuscript. Here's to answer and respond to your questions: 1. Introduction • We believe that there is no prior investigation linked to this topic ever done before in Indonesia. We added this information in the second and last paragraphs of Introduction 2. Methods • Yes, the bacterial culture examination was done on the suspicious patients only. Please refer to the last sentence of the paragraph 8 in the Discussion section. 3. Results • We have enclosed the leucocyte and procalcitonin data of the patients on the table 1 on Result section 4. Discussion • In the study to come, further investigation can probably be done in the low health-care setting, also bigger concerns on under-diagnosed bacterial co-infection of elderly and immunocompromised patients can be taken into consideration. we added this sentence on the last paragraph of the discussion. • The implication of the study is that we find the bacterial culture examination necessary in the COVID-19 patients management since the bacterial co-infection might worsen the clinical outcome. Therefore this examination should be conducted when the patient shows signs of infection during the intensive care. We added this sentence in the second paragraph of the introduction. Furthermore, we also appreciate your suggestions in order to improve the final quality of this manuscript. We are currently working on the minor revisions, proof-reading, and going to submit the latest version it promptly” Hope this will meet all the requirement and we will receive the final pdf proof soon. Thanks in advance, Tri Pudy Asmarawati and research team" } ] }, { "id": "81317", "date": "23 Mar 2021", "name": "Erni J. Nelwan", "expertise": [ "Reviewer Expertise tropical medicine and infectious disease" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nA well written article and proper data collection to support the conclusion. From a LMIC like Indonesia, this article shares important findings, since the co-bacterial infection is not more than 20%, higher than reported in IDSA data but lower compared to what had been expected in clinical practice among Indonesian doctors.\n\nAuthors might add value by putting a highlight on what is the clinical appearance of the co-bacterial infected vs. the non co-bacterial infected for readers to apply in daily practice.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-113
https://f1000research.com/articles/10-2/v1	05 Jan 21	{ "type": "Research Article", "title": "In silico analysis of cross reactivity among phospholipases from Hymenoptera species", "authors": [ "Yuliana Emiliani", "Andrés Sánchez", "Marlon Munera", "Jorge Sánchez", "Dilia Aparicio", "Yuliana Emiliani", "Marlon Munera", "Jorge Sánchez", "Dilia Aparicio" ], "abstract": "Background: Phospholipases are enzymes with the capacity to hydrolyze membrane lipids and have been characterized in several allergenic sources, such as hymenoptera species. However, cross-reactivity among phospholipases allergens are little understood. The objective of this study was to determine potential antigenic regions involved in cross-reactivity among allergens of phospholipases using an in silico approach. Methods: In total, 18 amino acids sequences belonging to phospholipase family derived from species of the order hymenoptera were retrieved from the UniProt database to perform phylogenetic analysis to determine the closest molecular relationship. Multialignment was done to identify conserved regions and matched with antigenic regions predicted by ElliPro server. 3D models were obtained from modeling by homology and were used to locate cross-reactive antigenic regions. Results: Phylogenetic analysis showed that the 18 phospholipases split into four monophyletic clades (named here as A, B, C and D). Phospholipases from A clade shared an amino acid sequences’ identity of 79%. Antigenic patches predicted by Ellipro were located in highly conserved regions, suggesting that they could be involved in cross-reactivity in this group (Ves v 1, Ves a 1 and Ves m 1). Conclusions: At this point, we advanced to the characterization of potential antigenic sites involved in cross-reactivity among phospholipases. Inhibition assays are needed to confirm our finding.", "keywords": [ "phospholipase", "Hymenoptera", "allergen", "in silico", "epitope", "cross-reactivity." ], "content": "Introduction\n\nAllergic diseases have become a public health problem; the genetic background of patients (atopy) and the environmental conditions are considered the cause of the increased risk to develop allergic diseases1. Exposure to allergens (typically harmless antigens in the environment) also promotes an immune response mediated by IgE. Over the last few years, species belonging to the order Hymenoptera have been characterized as potential allergenic sources. They represent a common source of sensitization, with more than 200,000 species including bees, wasps, and ants. Most of the member of this order are cosmopolitan species, but some of them have an endemic distribution with a capacity of sensitization , like the Bombus sp. located more frequently in central and northern Europe, whereas the yellowjacket (YJ) (Vespula spp.) and honeybee (HB) (Apis mellifera) are allergenic sources in North America. Other wasps such as Polistinae are found in southern Europe and America2–4.\n\nAllergic immune response from hymenopteran allergens has been studied in detail due to a high incidence of sting reactions to these insects. Approximately, 9.2% to 28.7% of the adult population is sensitized to the venom of hymenopterans5. Allergic response to Hymenoptera venom is one of the leading causes of anaphylaxis worldwide with a frequency of 27%, as compared to medications (41%) and foods (20%)6,3. Molecular, structural, and immunological characterization of hymenopteran venom allergens is advanced, in total 75 allergens from 31 different species have been explored, and since phospholipases are a family of allergens with clinical and biological relevance, some proteins belonging to this order such as hyaluronidase and antigen V are also considered relevant to sensitization to this allergenic2,7,8. Exposure to hymenoptera allergens is associated with bites and stings; it is considered that 56.6–94.5% of the general population have been bitten at least once in their life9.\n\nPhospholipases (PLA) are a major component of the venom of these species, representing 75% of the total mass of the poison and has been characterized as one of the main allergens in Hymenoptera10. They can be found in venoms from other arthropods such as chelicerates, in the venom of ophidians, as well as in different tissues of mammals such as pancreatic juice, synovial arthritic fluid. The superfamily includes 42 groups distributed in four types: A, B, C, and D11.\n\nPhospholipases belonging to class A split into two groups: class A1 hydrolyzes the phospholipid ester bond between the first acyl and glycerol (1 acyl-SN-glycerol phosphate), while class A2 hydrolyzes the bond between the second acyl and glycerol (2 Acyl-SN-glycerol phosphate). They are a family of enzymes with different molecular weights, PLA1 has a molecular weight of 28 KDa, while PLA2 are classified as high molecular weight cytosolic PLA2 (40–85 kDa) and low molecular weight secretory PLA2 (14–18 kDa) with the capacity to hydrolyze fatty acids that are present on the cell membrane and other types of lipophilic substances or participate in the mechanism of regulation of gene expression through the production of free fatty acids, from which cyclooxygenases synthesize prostaglandins12–14.\n\nThe structure, function, mechanisms, and cell signaling of PLA have been extensively studied; one important aspect of PLA is their capacity to induce allergic responses. Several epitopes involved in the co-sensitization of some PLA that share structural homology and identity have been studied; this suggests a potential role in cross-reactivity. However, this is little understood and studies are needed to complement what has been reported. The aim of this work was to explore cross-reactivity and antigenicity of allergenic PLA using an in silico approach, using bioinformatics tools, where we identified several antigenic regions that may be involved in cross-reactivity among phospholipases.\n\nToday it is evident how the use of bioinformatics tools for science has grown; it is considered the first step to carry out experimental studies because they create a functional prediction. Understanding and predicting an individual clinical cross-reactivity to allergens is key to better management, treatment, and progression of new therapies for allergy to Hymenoptera; prediction can be performed by methods for the identification and computational mapping of specific IgE epitopes or epitopes reported in the Immune Epitope Database and Analysis Resource, which can help identify the areas that may be affecting patients’ health. Various studies have carried out on this methodology for predicting food allergen epitopes15–17.\n\nThe in silico methodology has been used in other work to report possible cross-reactivity based on proteins in studies of structural or functional homology, through bioinformatics tools18.\n\n\nMethodology\n\nThe amino acid sequences of phospholipases type A (A1 and A2) from 18 Hymenoptera species were selected according to the allergenic capacity reported. The sequences were obtained from the UniProt database (see Table 1 for a list of accession numbers). All Allergens that were reported in the WHO/IUIS Allergen Nomenclature Sub-Committee with a complete sequence were used. We did not include incomplete sequences for analysis. Three sequences are not reported as allergenic but were chosen to observe the differences in identity and the structures of several phospholipases. The identity degree among phospholipases was determined using the PRALINE web server. The parameters to perform the alignment were configured to use BLOSUM62 as the exchange matrix. The interactions used were 3 with an E value of 0.001.\n\nThe name of the allergen, source, and type of phospholipases and Uniprot code are detailed.\n\nThe Molecular Evolutionary Genetic Analysis (MEGA) program, version X was used to obtain phylogenetic trees, using the method of maximum parsimony of the taxa with the support of Bootstrap with 1000 repetitions as a measure of reliability and robustness under the assumption of a minimum evolution. In the topology, this model uses a comparative matrix to find the similarity between the amino acids of 18 sequences to establish the evolutionary proximity between the species. The matrix was constructed with all the amino acid sequences of the phospholipases recovered from the UniProt database and reported to the WHO/IUIS. Therefore, the more positive identity values found between the sequences, the greater their relationship will be, and the closer they will be located in the tree. All empty spaces were eliminated (complete deletions). From the global comparison and the homologies, the sum of the length of the branches (SBL) will be presented, which will determine the number of nodes and their position, including the \"groups\" of the evolutionarily closest sequences. Phylogenetic sub-analyses were carried out in order to identify the degree of identity of the groups formed. The alignment for phylogenetic analysis was carried out using CLUSTAL W, which performs alignments. The parameters to perform the multiple alignment were configured to use gap opening penalty of 10.00 and gap extension penalty of 0.20, and the divergent cutoff delay was 30%.\n\nThe phospholipases with 3D structures not reported in the Protein Data Bank were obtained by modeling based on homology using the SWISS-MODEL server. Quality was evaluated by means of several tools, including the Ramachandran charts, WHATIF, the QMEAN4 index (The Qualitative Analysis of Energy Analysis) using ProSA-web and the SWISS-MODEL server. The results were expressed as a number between 0 and 1. Higher numbers indicate higher reliability and energy values (force field GROMOS96). ElliPro tools were used to predict lineal and discontinues epitopes on a representative phospholipase for group. Residues with larger scores are associated with greater solvent accessibility. Only residues with a score > 0.7 were selected.\n\n\nResults\n\nWe selected 18 sequences of allergenic phospholipases and three not allergenic to include in the analysis with 361 positions in the final dataset. The sequences were derived from several biological sources: five from bees, six wasps, three ants, and three sources not described as an allergen, mosquito, spider, and scorpion. The allergens of bees and wasps belong to group 1 and the ants to groups 1 and 2 (Table 1).\n\nThe phylogenetic tree had a consistency index of 0.857256 with a retention index of 0.779682 and a composite index of 0.683688 (0.668387) for all sites and parsimony-informative sites. A closed relationship among phospholipase allergens as shown, formed four nodes with a high phylogenetic relationship among them (Figure 1). According to the tree, group A grouped three phospholipase A1 all belonging to the Vespula genus (Ves v 1, Ves m 1, and Ves s 1). This group presents the greatest relationship among the groups with the closest distance between branches. Meanwhile, group B contains the highest number of phospholipases A2 phylogenetically related, including allergens of the Bombus and Apis genera (Bom p 1, Bom t 1, Api m 1, Api c 1) and two non-allergic phospholipases from Parasteatoda tepidariorum (Common house spider) and Centruroides hentzi (Hentz striped scorpion). Group C included four proteins, three of them from ants belonging to Solenopsis gender (Sol i 1, Sol i 2, Sol s 2) and one belonging to the mosquito C. quinquefasciatus. In group D we found all the wasp allergens that belong to the genus Polistes (Pol a 1, Pol d 1), P. Paulista (Poly p 1), and D. maculate (Dol m 1) and V. crabro (Vesp c 1).\n\nThe formation of fourth clades (A–D) with the highest degree of identity is observed (79% for clade A). The evolutionary history was inferred using the Maximum Parsimony method. The most parsimonious tree with length equal to 1462 is shown.\n\nMultiple alignments of the phospholipases of the different groups obtained from the phylogenetic analyzes were made. We built four 3D models of the 18 phospholipases Ves s 1, Sol i 1, Culex quinquefasciatus and Centruroides hentzi. The remaining proteins were reported on the UniProt database. We considered structures for better visibility of antigenic patches, the parameters for structural quality control for homology models are found in Table 3. To compare the ElliPro results, we chose the main antigen patches with a score higher than 0.7 and more than three residues, taking as reference the epitope of one phospholipase of each group; group A: Ves m 1; group B: Bom p 1; group C: Sol i 1; Group D: Pol d 1 (Table 2). The constitutional antigenic patches are shown in Figure 2.\n\nThe QMEAN4 index has a score is between the range (0-1) that indicates good quality in the model and the GQME index is expressed as a number between 0 and 1. Higher numbers indicate greater reliability.\n\nThe Sol I 1 structure was obtained by homology, using the Vespid basal is sequence as a template (34.32% de identical; QMEAN -5, 45).\n\nPhospholipases from group A had a shared identity of 79% between their amino acid sequences (Figure 3). A total of 704 residues were identified and conserved among the phospholipases analyzed, and for these group, we used Ves m 1 to identify the possible epitopes. We found three common linear antigenic patches and two constitutive antigenic patches with a score greater than 0.7.\n\nUnconserved sequence are shown with blue color and high conserved sequence with red color. Moderately conserve sequence are showed with green and orange color. The alignment score was 14,674 with a total of 704 identities residues. The percent sequence identity was 79%.\n\nGroup B shares an identity of 35% between their amino acid sequences but when we exclude Api c 1, the identity increases to 64%. In total, 259 identical residues among the sequences were found. We found and included three linear epitopes and two discontinues antigenic patches in Bom p 1 with a score >0.7. (Figure 4).\n\nThree sequences were studied with a total of 439 residues. Unconserved sequence are shown with blue color and high conserved sequence with red color. Middle conserve sequence are showed with green and orange color. A total of 251 residues were identities. The percent sequence identity was 64%.\n\nGroup C, which includes allergens from ants, showed the lowest identity, with only 23% and the highest number of gaps (600 residues missing). Sol i 1 was the protein furthest away from any of the Hymenoptera allergens and appears to be closely related with wasps’ allergens. No common antigenic patches were detected; however, Sol l 1 presents an interesting antigenic patch with 46 residues and a score of 0.711.\n\nFor group D, 1916 residues exhibit an identity among the five sequences of allergens. This group exhibit a high identity of 64%, the second highest after clade A. For the identification of antigen patches in this group, we used Pol d 1 finding four linear epitopes but only one continuous epitope with a valid score (Figure 5).\n\nFive sequences were studied with a total of 1564 residues. Unconserved sequences are shown with blue color and highly conserved sequence with red color. Middle conserve sequence are shown with green and orange colors. The percent sequence identity was 64% with a total of 1916 Aidentities residues.\n\n\nDiscussion\n\nPhospholipases A1 and A2 are allergens of insects, which provide a diagnostic benefit for the differentiation of genuine cross-reactivity sensitization. However, the cross-reactivity of this group of allergens has scarcely been holistically explored. In this study, we were able to predict those possible antigenic regions that could explain the cross-reactivity of phospholipases in Hymenoptera through in silico analyses.\n\nThe 18 amino acid sequences of the allergens were aligned, and a phylogenetic analysis was carried out which yielded four monophylogenetic groups (A, B, C, D). Group A yielded the highest degree of identity among their amino acid sequences (79%). All the allergens of this group belong to the Vespula genus, one of the most studied sources of wasp allergens7,19. In group B (Bom p 1, Bom t 1, Api m 1, Api c 1) two analyses were conducted, the first with the presence of the Api c 1 allergen where a degree of identity of (35%) was found and the second without the allergen, where we found a higher degree of identity at (64%). This showed that the alignment of these three species could explain a possible cross-reactivity. Group C (Pol a 1, Pol d 1, Poly p 1, Vesp c 1, Dol m 1.02) showed a level of identity of (64%). However, analysis of conserved and affected residues showed that Group A shares three antigenic regions that could contribute to their cross-reactivity.\n\nIgE against cross-reactive carbohydrate determinants (CCD) is one of the main causes of double positivity and is present in most hymenopteran venom allergens with more frequency in venom from HB and YJ in patients that are allergic to insect bites20. The prevalence of this allergen has been described in more than 20% of patients allergic to honeybee venom; approximately one of four HB poisons and one of 10 YJ venom allergens have been found to be CCDsIgE-positive. The PLA2 structure contains the insect CCDs that are specified by the presence of a 3-core α-1 fucose21. Insect CCD causes 69% at 75% double positive test results for HBV and YJV during allergy diagnosis20–22. Hemmer et al. propose that the Radio Allergo Sorbent Test (RAST) results to OSR pollen appear to be a simple and practicable measure to detect sugar specific IgE in individual sera. This could be useful to discriminate between patients who cross react through CCD and doubly sensitized patients who may require immunotherapy with two poisons.\n\nCurrently, CCD-free allergens have been known to allow cross reactivity between proteins to be found without having a double positivity. Ves v 1, Api m 1, Dol m 1, Pol d 1 are allergens that lack cross-reactivity based on CCD and allow diagnoses without interference19,23,24. However, it should be clarified that these are mostly of recombinant origin because in its purified natural form possess CCD; for example, Api m 1 of natural origin has CCD and makes diagnosis difficult24. On the other hand, Sol i 1 is the only PLA1 hymenopteran venom known to have CCD, which could make the specific diagnosis of fire ant allergy difficult25.\n\nResearch on the allergenic capacity of Hymenoptera allergens has been characterized by individualized studies, with Api m 1, Sol i 1, Pol d 1, Ves m 1 among those most studied so far, but the possible cross-reactivity between phospholipase allergens A1 and A2 has not been holistically evaluated2,24,26. No cross-reactivity between A. mellifera, S. invicta and V. vulgaris was detected, which supports our results, since there was no relationship between these allergens. However, when analyzed along with other allergens, it was observed that a certain degree of identity is maintained between these two proteins, suggesting a possible cross reactivity without CCD.\n\nGroup A (Ves m 1, Ves s 1 and Ves v 1) being the most representative, the cross reactivity between Vespula spp. is strong due to the similarities in the composition of the poison and the structure of the individual allergens27. Different studies evaluate the identity of the yellow jackets; for example, a 1996 study reported that Ves v 1 had 95% identity with Ves m 1 and both yellow jacket phospholipases have about 67% sequence identity with the hornet protein Dol m 17. Other authors demonstrated that Ves v 1 also shows an identity of 54% with Poly p 1, it being the lowest among the allergens studied and a study carried out in Spain with 59 previously diagnosed allergic patients with an allergy to vespids found that there could be a double sensitization between Ves v 1 and Pol d 1 because in 31% of patients they could not be clearly defined as sensitized only to Vespula or Polistes28,29. Consequently, the different Vespula poisons react strongly in a crossed manner, which would explain the high degree of identity found in the study (Group A (79%)). Of the three proteins, only Ves v 1 has been described as a CCD allergen, showing that this interaction between the Vespula phospholipases could be CCD-independent and related only by protein structure19. The quaternary structure of the three Vespula phospholipases is also very similar, suggesting the possibility of present both linear and conformational epitopes (Figure 6A). Therefore, we suggested that fragment inhibition studies be carried out to identify the possible antigenic peptide described in this study.\n\n(A) Consisting of Ves v 1 in green, Ves m 1 in orange and Ves s 1 in blue. (B) Consisting of Api c 1, Api m 1, Bom p 1, Bom t 1, Centruroides hentzi and Parasteatoda tepidariorum, but we only found structural homology in Api m 1 of orange color, Bom p 1 of yellow color and Bom t 1 of red color. (C) Consisting of Sol i 1, Sol i 2, Sol s 1, and Culex quinquefasciatus but we only found structural homology in Sol i 2 of a magenta color, Sol s 1 of blue color. (D) Consisting of Pol a 1 of green color, Pol d 1 of yellow color, Poly p 1 of blue color, Dol m 1 of cyan color and Vesp c 1 of red color.\n\nGroup B showed a degree of identity of 35%, however, in the analysis, we found that if we performed the alignment without the Api c 1 allergen, the degree of conservation between Api m 1, Bom p 1 and Bom t 1 increased to 64%. So far, we have found no more information about the possible cross reactivity in these allergens. In this group, Api m 1 is the most characterized allergen; It contains the cross-reactive carbohydrate (CCD) determinants of insects that are defined by the presence of a 3-core α-1 fucose30.\n\nFor years, the detection of Api m 1 CCD challenges the differentiation of HB and YJ allergy. However, in vitro detection of immunoreactive sIgE from these insects showed double positivity in up to 59% of the patients24. PLA2s possess important venom allergens in other members of the genus Apis and Bombus that have been shown to have homology. A. cerena (Api c 1) have been little explored but have been described as having high identity levels with other phospholipases, like A. mellifera (95%)26. In our study, we observed that when comparing the sequences of these phospholipases with those of the genus Bombus, that identity was not preserved since the identity we found was very low and when excluding it from the alignment, the sequences were more conserved31. Studies conducted on the genus Bombus found that the primary sequences of Bom t 1 and Api m 1 have an identity of 53% and their three-dimensional structures show conserved low protein surfaces32. However, the allergens selected from group B in our study showed a high conservation and structural homology leading to possible cross-reactivity (Figure 6B).\n\nAs for Group C, we highlight that it was the only group that included phospholipases A1 and A2 in the clade, so a low identity was expected. We found that the ant phospholipases Sol i 1, Sol i 2 and Sol s 2 showed a degree of alignment identity with the other phospholipases in the primary sequences of 23%. This low identity is not enough to explain cross-reactivity in silico, even though allergen Sol i 1 has been extensively analyzed and other studies suggest that it may have a possible reactivity with the Centruroides species33,34.\n\nThe phylogenetic analyzes reported in this study revealed that Sol i 1 is the most divergent member among the currently identified hymenopteran venom group PLA1. As noted, Sol i 1 is in a group (group C) completely isolated from the clade consisting of wasp allergenic PLA1 (group A) and showed no structural homology (Figure 6C). Furthermore, in multiple alignments, the fire ant exhibits the lowest level of sequence identity. However, studies have shown cross-reactivity between Sol i 1 and its wasp counterparts with amino acid sequence identity levels of 38% with Ves m 1, 36% with Ves v 1, 40% with Dol m, 1.35% with Pol d. 1.36% with Poly p 135. However, a recent study suggests that peptide-based cross-reactivity between Sol i 1 and PLA1 of Polistinae wasps does not occur because the alignments and the phylogenetic and structural analyzes showed that it is an allergen further from its counterparts, in addition to possessing the lowest level of identity among the sequences studied, with 36%, and the highest RMSD value with 0.17229.\n\nSeveral works have attempted to demonstrate cross-reactivity between A1 phospholipases29,36. The cross-reactivity based on PLA1 of the venoms of eight hymenoptera was analyzed and it was described that the identity of the primary sequence of Poly p 1 was conserved in 36% with Sol i 1, 74% with Pol d 1 and 71 % with Pol a 1. In our study no relationship was found between Poly p 1 with Sol i 1. However, group D, where we found the different species of Polistes (Pol a 1 and Pol s 1), Poly p 1, Dol m 1 and Vesp c 1, showed a high degree of identity of 64% and structure homology (Figure 6D), enough to explain cross-reactivity29. An attempt was made to look for cross reactivity between Dol m 1, Ves v 1 and pol a 1 with mice; partial cross-reactivities in the T-cell epitopes of homologous vespid allergens was found, which supports our findings7,29,36.\n\nOf the species chosen, three non-allergenic phospholipases (Centruroides hentzi, Parasteatoda tepidariorum, Culex quinquefasciatus) were taken to adjust the phylogenetic analysis, so as the results were produced, we observed that these phospholipases separated into two clades showing some affinity for some phospholipases allergens.\n\nA study identified allergens in the venom of common striped scorpions. Eleven patients with scorpion venom allergy were assessed, where four patients had a history of anaphylaxis (with positive skin test responses) to imported fire ant venom (IFA) and at least two other had a history of large local reactions, suggesting that there could be a cross reactivity between proteins of these insects; this association would be clinically relevant29. This shows that despite not being described as allergens, it is necessary to carry out studies to verify their capacity to trigger sensitization.\n\nBioinformatic studies are high impact tools of great importance. Currently they are recognized as the first step to conducting an investigation, since they are in silico analyzes that facilitate a possible approximation to expected results, allow predictions or models, and serve as the basis for the emergence of large projects. In our study, we show possible antigenic regions involved in cross-reactivity between phospholipases A1 and A2, based on what was found with the use of in silico analysis we can say that they are proteins with a high degree of identity and that three antigenic regions were found, which would explain possible co-sensitization.\n\n\nConclusion\n\nPotential antigenic sites were identified for the generation of cross-reactivity between the phospholipases analyzed in this study. The identity between these proteins of different species is relatively high, which shows that cross-reactivity between them is possible and their frequency in most cases can be high. These studies support diagnostic testing by component studies for venom allergy and the need to carry targeted mutagenesis tests is important to confirm their relevance in the allergenic capacity of phospholipases.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "References\n\nGilles S, Akdis C, Lauener R, et al.: The role of environmental factors in allergy: A critical reappraisal. Exp Dermatol. 2018; 27(11): 1193–200. PubMed Abstract \| Publisher Full Text\n\nJakob T, Müller U, Helbling A, et al.: Component Resolved Diagnostics for Hymenoptera Venom Allergy. Curr Opin Allergy Clin Immunol. 2017; 17(5): 363–72. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJakob T, Rafei-shamsabadi D, Spillner E, et al.: Diagnostics in Hymenoptera venom allergy: current concepts and developments with special focus on molecular allergy diagnostics. Allergo J Int. 2017; 26(3): 93–105. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlank S, Neu C, Hasche D, et al.: Polistes Species Venom Is Devoid of Carbohydrate-Based Cross-Reactivity and Allows Interference-Free Diagnostics. J Allergy Clin Immunol. 2013; 131(4): 1239–42. PubMed Abstract \| Publisher Full Text\n\nBilò BM, Floriano B: Epidemiology of Insect-Venom Anaphylaxis. Curr Opin Allergy Clin Immunol. 2008; 8(4): 330–37. PubMed Abstract \| Publisher Full Text\n\nOllert M, Simon B: Anaphylaxis to Insect Venom Allergens: Role of Molecular Diagnostics. Curr Allergy Asthma Rep. 2015; 15(5): 26. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKing TP, Lu G, Gonzalez M, et al.: Yellow Jacket Venom Allergens, Hyaluronidase and Phospholipase: Sequence Similarity and Antigenic Cross-Reactivity with Their Hornet and Wasp Homologs and Possible Implications for Clinical Allergy. J Allergy Clin Immunol. 1996; 98(3): 588–600. PubMed Abstract \| Publisher Full Text\n\nHoffman DR: Allergens in Hymenoptera venom. XXV: The amino acid sequences of antigen 5 molecules and the structural basis of antigenic cross-reactivity. J Allergy Clin Immunol. 1993; 92(5): 707–16. PubMed Abstract \| Publisher Full Text\n\nBlank S, Stephanie H, Jaeger T, et al.: Prevalence of Hymenoptera Venom Allergy and Sensitization in the Population-Representative German KORA Cohort. Allergo J Int. 2019; 28(6): 183–91. Publisher Full Text\n\nFalcó, SN, Ferré Ybarz L: Hipersensibilidad a Veneno de Himenópteros. Protoc Diag Ter Pediatr. 2013; 1: 135–79. Reference Source\n\nSpillner E, Blank S, Jakob T, et al.: Hymenoptera allergens: from venom to \"venome\". Front Immunol. 2014; 5: 77. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBurke JE, Edward AD: Phospholipase A2 structure/function, mechanism, and signaling. J Lipid Res. 2009; 50 Suppl(Suppl): S237–42. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRock CO, Jackowski S, Cronan JE Jr, et al.: Chapter 2 Lipid Metabolism in Prokaryotes. New Comprehensive Biochemistry. 1996; 31. Publisher Full Text\n\nGomez F, Vandermeers A, Vandermeers-Piret MC, et al.: Purification and Characterization of Five Variants of Phospholipase A2 and Complete Primary Structure of the Main Phospholipase A2 Variant in Heloderma Suspectum (Gila Monster) Venom. Eur J Biochem. 1989; 186(1–2): 23–33. PubMed Abstract \| Publisher Full Text\n\nNugraha R, Kamath SD, Johnston E, et al.: Conservation Analysis of B-Cell Allergen Epitopes to Predict Clinical Cross-Reactivity between Shellfish and Inhalant Invertebrate Allergens. Front Immunol. 2019; 10: 2676. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDall F, Pavkov-Keller T, Zangger K, et al.: Structure of allergens and structure based epitope predictions. Methods. 2014; 66(1): 3–21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMaleki SJ, Teuber SS, Cheng H, et al.: Computationally predicted IgE epitopes of walnut allergens contribute to cross-reactivity with peanuts. Allergy. 2011; 66(12): 1522–29. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMarlon M, Andres S, Jorge S, et al.: In Silico Analysis of Cross Reactivity between Lipocalin of Domestic Animals*. Open Journal of Immunology. 2018; 08(04): 97–106. Publisher Full Text\n\nSeismann H, Blank S, Cifuentes L, et al.: Recombinant phospholipase A1 (Ves v 1) from yellow jacket venom for improved diagnosis of hymenoptera venom hypersensitivity. Clin Mol Allergy. 2010; 8: 7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHemmer W, Focke M, Kolarich D, et al.: Antibody Binding to Venom Carbohydrates Is a Frequent Cause for Double Positivity to Honeybee and Yellow Jacket Venom in Patients with Stinging-Insect Allergy. J Allergy Clin Immunol. 2001; 108(6): 1045–52. PubMed Abstract \| Publisher Full Text\n\nBlank S, Michel Y, Seismann H, et al.: Evaluation of Different Glycoforms of Honeybee Venom Major Allergen Phospholipase A2 (Api m 1) Produced in Insect Cells. Protein Pept Lett. 2011; 18(4): 415–22. PubMed Abstract \| Publisher Full Text\n\nMüller UR, Johansen N, Petersen AB, et al.: Hymenoptera venom allergy: analysis of double positivity to honey bee and Vespula venom by estimation of IgE antibodies to species-specific major allergens Api m1 and Ves v5. Allergy. 2009; 64(4): 543–48. PubMed Abstract \| Publisher Full Text\n\nMüller UR: Recombinant Hymenoptera Venom Allergens. Allergy. 2002; 57(7): 570–76. PubMed Abstract \| Publisher Full Text\n\nMüller U, Schmid-Grendelmeier P, Hausmann O, et al.: IgE to recombinant allergens Api m 1, Ves v 1, and Ves v 5 distinguish double sensitization from crossreaction in venom allergy. Allergy. 2012; 67(8): 1069–73. PubMed Abstract \| Publisher Full Text\n\nPerez-Riverol A, Miehe M, Jabs F, et al.: Venoms of Neotropical Wasps Lack Cross-Reactive Carbohydrate Determinants Enabling Reliable Protein-Based Specific IgE Determination. J Allergy Clin Immunol. 2018; 141(5): 1917–1919.e1. PubMed Abstract \| Publisher Full Text\n\nLi JH, Zhang CX, Shen lR, et al.: Expression and Regulation of Phospholipase A2 in Venom Gland of the Chinese Honeybee, Apis Cerana Cerana. Arch Insect Biochem Physiol. 2005; 60(1): 1–12. PubMed Abstract \| Publisher Full Text\n\nWicher K, Reisman RE, Wypych J, et al.: Comparison of the Venom Immunogenicity of Various Species of Yellow Jackets (Genus Vespula). J Allergy Clin Immunol. 1980; 66(3): 244–49. PubMed Abstract \| Publisher Full Text\n\nMonsalve RI, Vega A, Marqués L, et al.: Component-Resolved Diagnosis of Vespid Venom-Allergic Individuals: Phospholipases and Antigen 5s Are Necessary to Identify Vespula or Polistes Sensitization. Allergy. 2012; 67(4): 528–36. PubMed Abstract \| Publisher Full Text\n\nPerez-Riverol A, Luís G, et al.: Phospholipase A1-Based Cross-Reactivity among Venoms of Clinically Relevant Hymenoptera from Neotropical and Temperate Regions. Mol Immunol. 2018; 93: 87–93. PubMed Abstract \| Publisher Full Text\n\nJakob T, Julian K, Blank S, et al.: Comparable IgE Reactivity to Natural and Recombinant Api m 1 in Cross-Reactive Carbohydrate Determinant-Negative Patients with Bee Venom Allergy. J Allergy Clin Immunol. 2012; 130(1): 276–78. PubMed Abstract \| Publisher Full Text\n\nHoffman DR, Jacobson RS: Allergens in Hymenoptera venom. XXVII: bumblebee venom allergy and allergens. J Allergy Clin Immunol. 1996; 97(3): 812–21. PubMed Abstract \| Publisher Full Text\n\nVan Vaerenbergh M, Debyser G, Smagghe G, et al.: Unraveling the Venom Proteome of the Bumblebee (Bombus Terrestris) by Integrating a Combinatorial Peptide Ligand Library Approach with FT-ICR MS. Toxicon. 2015; 102: 81–88. PubMed Abstract \| Publisher Full Text\n\nMore D, Nugent J, Hagan L, et al.: Identification of Allergens in the Venom of the Common Striped Scorpion. Ann Allergy Asthma Immunol. 2004; 93(5): 493–98. PubMed Abstract \| Publisher Full Text\n\nNugent JS, More DR, Hagan LL, et al.: Cross-Reactivity between Allergens in the Venom of the Common Striped Scorpion and the Imported Fire Ant. J Allergy Clin Immunol. 2004; 114(2): 383–86. PubMed Abstract \| Publisher Full Text\n\nHoffman DR, Sakell RH, Schmidt M, et al.: Sol i 1, the phospholipase allergen of imported fire ant venom. J Allergy Clin Immunol. 2005; 115(3): 611–16. PubMed Abstract \| Publisher Full Text\n\nPerez-Riverol A, Musacchio Lasa A, Aparecido Dos Santos-Pinto JR, et al.: Insect Venom Phospholipases A1 and A2: Roles in the Envenoming Process and Allergy. Insect Biochem Mol Biol. 2019; 105: 10–24. PubMed Abstract \| Publisher Full Text" }	[ { "id": "76669", "date": "18 Jan 2021", "name": "Anna Pomés", "expertise": [ "Reviewer Expertise Allergen structure/function and antigenic determinants" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGeneral comments: The manuscript by Emiliani et al. reports an in silico analysis of cross-reactivity among phospholipases from Hymenoptera species. Four groups or clades are identified and further analyzed by sequence alignments and epitope prediction. However, the potential IgE cross-reactivity between pairs of proteins within and between different clades are not clear from the analysis. The discussion needs to be revised to explain better their results compared to clinical observations reported in the literature.\nFirst paragraph from results: From Table 1 it seems that 15 allergenic and 3 non allergenic sequences were selected. The way it is phrased it seems as if 18 allergenic sequences were selected. The numbers of species do not add up to 18 (5 bees, 6 wasps, 3 ants, 3 other = 17). Are yellow jackets and hornets considered wasps? If so there should be 8 wasps, and 4 bees.\nTable 3: The range 0-1 indicated for QMEAN4 index in the Legend to table 3 does not correspond to the range of values -6.49 to 0.23 in the Table. “has a score is” needs correction.\nThe authors should explain what do they mean by “constitutional” or “constitutive” antigenic patches (page 5). Do the authors mean “conformational or discontinuous epitopes”? (not “discontinues” as in page 6).\nLegend to figure 2: Sol i 1 (not Sol I 1). “the Vespid basal is sequence” does not seem correct.\nPage 5, 3rd paragraph: how can 704 residues be conserved among 337 residues per sequence (in the alignment of Figure 3). The authors should explain where the number 704 comes from. Similarly, the same applies to 259 and 1916 residues from sequences that are shorter in Figures 4 and 5, respectively. Usually, percent of identity between two sequences is a better way to express homology than saying the total residues that were conserved for all the sequences (this is what the authors seem to be presenting). A better way that the authors could use to show homologies among several sequences, is an Percent Identity Matrix, which shows all the percentages of identity between pairs of proteins.\nSol i 1 in page 6, second paragraph.\nLinear and continuous epitopes are the same. In page 8, first paragraph: do the authors mean to use linear and continuous in the same line?\nDiscussion, end of paragraph 2: Do groups A and D share antigenic areas? (what are “affected residues”?). If so, the explanation is not clear, and not shown in the results. Maybe an overlay of a representative molecule from each of the two groups showing the areas that are shared in green could be helpful in the results section to illustrate this (and blue and yellow areas in the respective molecules).\nDiscussion, third paragraph: “double positivity” needs to be explained (69% at 75% is not clear). What is OSR? (spell out).\nThe discussion is hard to follow, maybe in part because it is not clear from the results if cross-reactivity is expected or not among different species. It might be interesting to have a table in the results section showing the 4 groups with their proteins (in first row and first column) and indicating if clinical cross-reactivity has been observed (also CCD presence or absence if it applies), next to expected cross-reactivities from the results.\nMinor comments:\nEnglish grammar needs revision. For example, in Abstract: “phospholipase allergens”, “18 amino acid sequences”, “shared an amino acid sequence identity”.\n\nSome terms need explanation for the readers to understand (for example: “empty spaces”, “length of the branches”, “nodes” in first paragraph, page 4).\n\nPage 3, 1st column, paragraph 2, line 14: “sensitization to this allergenic.” Needs to be completed (allergenic source?).\n\nPage 3, second column, 3rd paragraph, line 10: sentence with “areas” is vague. Does “area” mean epitopes or something else? Line 11: “Various studies have been carried out…”\n\nPage 5, first paragraph: P. paulista. Other species names should be in italics as well all over the manuscript (i.e. Legend to Figure 6).\n\nPage 5, 1st paragraph, last line: analyses.\n\nLegends to Figures 3, 4 and 5 need corrections (highly conserved, middle conserved sequences, were identified).\n\nVerb “Consisting” can be removed from Legend to figure 6.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "6436", "date": "29 Mar 2021", "name": "Andrés Sánchez", "role": "Author Response", "response": "Anna Pomés. Indoor Biotechnologies, Inc., Charlottesville, United States. Dear reviewer. We have addressed all the suggestions, below are the comments. Answer at the comments: 1. First paragraph from results: From Table 1 it seems that 15 allergenic and 3 no allergenic sequences were selected. The way it is phrased it seems as if 18 allergenic sequences were selected. The numbers of species do not add up to 18 (5 bees, 6 wasps, 3 ants, 3 other = 17). Are yellow jackets and hornets considered wasps? If so there should be 8 wasps, and 4 bees. Answer: First paragraph of the results an error is evidenced, because the correct phrase is how you put it. We selection 15 sequences allergenic and 3 nor allergenic. The sum of the species was organized bad, the really are 2 bees, 2 bumblebee, 6 wasp, 2 hornets, 3 ant and 3 no allergenic, respectively. 2. Table 3: The range 0-1 indicated for QMEAN4 index in the Legend to table 3 does not correspond to the range of values -6.49 to 0.23 in the Table. “has a score is” needs correction. Answer: the global score is originally in a rank [0, 1] being one a good. Default, the transformed in Z score from relate then to what we would expect from X ray structure of high resolution. 3. The authors should explain what do they mean by “constitutional” or “constitutive” antigenic patches (page 5). Do the authors mean “conformational or discontinuous epitopes”? (not “discontinues” as in page 6). Answer: we accept the observation and proceed to change constitutive antigenic parch from conformational epitopes in the second version. 4. Legend to figure 2: Sol i 1 (not Sol I 1). “the Vespid basal is sequence” does not seem correct. Answer: it was correct the name of the antigen: Sol I 1, both in the legend as in the paragraph 6. 5. Page 5, 3rd paragraph: how can 704 residues be conserved among 337 residues per sequence (in the alignment of Figure 3). The authors should explain where the number 704 comes from. Similarly, the same applies to 259 and 1916 residues from sequences that are shorter in Figures 4 and 5, respectively. Usually, percent of identity between two sequences is a better way to express homology than saying the total residues that were conserved for all the sequences (this is what the authors seem to be presenting). A better way that the authors could use to show homologies among several sequences, is an Percent Identity Matrix, which shows all the percentages of identity between pairs of proteins. Answer: the PRALINE tool used for the alignment shows the number of residues taken into account for the analysis, what was done was to calculate how many residues of that total were conserved in those analyzed sequences. Thus, in figure 3, 934 of which 704 were conserved were taken into account in the analysis, this figure corresponds to 79% of the total residues and similarly applies to residues 259 and 1916 of figures 4 and 5. The matrix Percentage identity is included in the second version. 6. Sol i 1 in page 6, second paragraph. Answer: corrected 7. Linear and continuous epitopes are the same. In page 8, first paragraph: do the authors mean to use linear and continuous in the same line? Answer: Yes, they are the same and for illustrative purposes the term Epitope lineal is used in the second version. 8. Discussion, end of paragraph 2: Do groups A and D share antigenic areas? (what are “affected residues”?). If so, the explanation is not clear, and not shown in the results. Maybe an overlay of a representative molecule from each of the two groups showing the areas that are shared in green could be helpful in the results section to illustrate this (and blue and yellow areas in the respective molecules). Answer: In our study, we did not find antigenic areas directly related to cross-reactivity between group A and D proteins. However, in the study carried out by Hoffman, et al. 2005 showed that there are levels of identity between the proteins of these groups and they also include Sol i 1 found in group C. Therefore we use this information to compare the results found. 9. Discussion, third paragraph: “double positivity” needs to be explained (69% at 75% is not clear). What is OSR? (spell out). Answer: the double possibility refers to possible cross reactivities caused by the recognition of different substances contained in the analyzed insects, for example; In the particular case of some bees and wasps, they present double cross-reactivity given by the carbohydrate cross-reaction determinants (CCD) and by phospholipases, as they are different substances but both are recognized in insects, they are called “double positivity”. The percentages show that 69 to 75% of the tests performed for bee venom and yellow jacket venom give double positivity due to the presence of carbohydrate cross-reaction determinants (CCD) and phospholipases, which confuses the diagnosis allergy. OSR: Oilseed rape This reference explain the concept of positivity double: - Hemmer, Wolfgang, et al. Antibody Binding to Venom Carbohydrates Is a Frequent Cause for Double Positivity to Honeybee and Yellow Jacket Venom in Patients with Stinging-Insect Allergy. pp. 1045–52, doi:10.1067/mai.2001.120013. 10. The discussion is hard to follow, maybe in part because it is not clear from the results if cross-reactivity is expected or not among different species. It might be interesting to have a table in the results section showing the 4 groups with their proteins (in first row and first column) and indicating if clinical cross-reactivity has been observed (also CCD presence or absence if it applies), next to expected cross-reactivities from the results. Answer: thanks for the suggestion, we will take it into account to carry out a review of the results, discussion in order to clarify the central ideas and make the information clearer for the reader. We make the suggested table for clarity of information. Minor comments: 1. English grammar needs revision. For example, in Summary: \"phospholipase allergens\", \"18 amino acid sequences\", \"share amino acid sequence identity\". Answer: English grammar will be reviewed. 2. Some terms need an explanation for readers to understand (for example: “empty spaces”, “length of branches”, “nodes” in the first paragraph, page 4). Answer: - The empty spaces are areas of different sequences where there is no relationship between their amino acids. - The branch length or branch distance refers to the relationship between the allergens exposed in the tree. The longer the branch length, the less related there is between the allergens and the shorter the greater the relationship. - Nodes is a synonym for groups or clades that make up the phylogenetic tree. 3. Page 3, first column, paragraph 2, line 14: \"sensitization to this allergen\". Must be completed (allergen source?). Answer: \"sensitization to this allergen\" is corrected by \"sensitization to hymenoptera allergens\" because the central idea revolves around the diversity of allergens of this order. 4. Page 3, second column, third paragraph, line 10: the sentence with “areas” is vague. Does \"area\" mean epitopes or something else? Line 11: \"Several studies have been carried out ...\" Answer: The term was corrected for conserved regions that refer to epitopes (these are the regions causing sensitization). What is mentioned on line 11 is support to show that other valid studies have been done. 5. Page 5, first paragraph: P. Paulista. Names of other species should also be italicized throughout the manuscript (ie, Legend to Figure 6). Answer: we italicize those names that were missing. 7. Legends in Figures 3, 4 and 5 need correction (highly conserved sequences identified, conserved in the middle). Answer: Conserved between the sequence with high identity 8. The verb \"Consistent\" can be removed from the legend to the figure Answer: the verb was removed." } ] }, { "id": "76667", "date": "03 Feb 2021", "name": "Jose F. Cantillo", "expertise": [ "Reviewer Expertise Immunology", "allergy", "recombinant allergens", "mosquito and house dust mite allergy", "somatic hypermutation and class switch recombination." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMajor comments: The manuscript presented by Emiliani et al. presents a theoretical analysis of cross-reactivity among Hymenoptera-derived phospholipases. 18 sequences were retrieved and splitted in 4 clades after phylogenetic analysis. Prediction of IgE-cross reactive epitopes was performed using a bioinformatic tool and multiple alignment. There are some considerations that the authors must address to provide a more solid argument, and further analysis must be performed.\nWhy did the authors used ElliPro server and not another one? Is there any advantage of this tool compared to others?\n\nPrediction of antigenic regions should be performed using different tools and individual analysis of accessibility, hydrophobicity, etc. And then compared the results to provide the “most probable” antigenic region.\n\nThe authors use the term “constitutional antigenic patches”. What does this mean? Is it a concept used by the bioinformatic tool? If so, they must explain what this is about.\n\nComparisons between aa sequences result in percentages of identity as a way to express the level of conservation or homology. However, proteins like phospholipases have variable molecular weights, which means that they aren’t similar in the whole length of their sequences. The authors must make clear that a portion of each protein is used to make the statements related to the identities and that the analysis is restricted to these areas.\n\nConsequently, was the homology model made for the whole Ves s 1, Sol I 1, C. quinquefasciatus and C. hentzi phospholipases as whole proteins or just a portion of them?\n\nIn the methods section, which templates were used by SWISS MODEL to predict the 3D model?\n\nHow was the superimposition of 3D structures presented in figure 5 performed?. The approach should be indicated in the methods.\n\nThe discussion is too long given the presented results. Sometimes the information provided is repetitive. For example, the second paragraph of the discussion is essentially previously mentioned in the results section and can be eliminated.\n\nThe third paragraph in the discussion section refers to carbohydrate determinants, which are not even superficially explored in this study. Although the observation referred to CCD can be mentioned in this manuscript, is not necessary to discuss this issue too much since there are no analysis for this matter in this paper.\n\nA similar evaluation of all the information provided in the discussion should be done. It is difficult to understand what the authors are trying to argue and how this is closely related to the actual results. A shortened and more precise discussion could greatly improve the manuscript.\nMinor comments:\nEnglish and grammar needs to be revised. Many sentences need to be re-written to better disclose the message that the authors are trying to say, for example:\nAllergic immune response from hymenopteran allergens has been studied in detail due to a high incidence of sting reactions to these insects…\n\nA closed relationship among phospholipase allergens as shown, formed four nodes with a high phylogenetic relationship among them…\n\nA better use of “comas” and “periods” must be done in order to improve the writing as well. For example: Molecular, structural, and immunological characterization of hymenopteran venom allergens is advanced, in total 75 allergens from 31 different species have been explored, and since phospholipases are a family of allergens with clinical and biological relevance, some proteins belonging to this order such as hyaluronidase and antigen V are also considered relevant to sensitization to this allergenic\n\nIn figure 3, what does “identities residues” mean?.\n\nIf a total of 704 residues were identified and conserved among the phospholipases, why is figure 3 showing around 330 only?.\n\nWe built four 3D models of the 18 phospholipases Ves s 1, Sol i 1, Culex quinquefasciatus and Centruroides hentzi… are you referring to “18” or “4”proteins?.\n\nFigures 3, 4 and 5 only show the multiple sequence alignments, but not the identified epitopes as the authors are pointing out. Please correct.\n\nIn group B (Bom p 1, Bom t 1, Api m 1, Api c 1) two analyses were conducted, the first with the presence of the Api c 1 allergen where a degree of identity of (35%) was found and the second without the allergen… why are you using parenthesis?\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "6437", "date": "29 Mar 2021", "name": "Andrés Sánchez", "role": "Author Response", "response": "Jose F. Cantillo Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL, USA Dear reviewer. We have addressed all the suggestions, below are the comments. Answer at the comments: 1. Why did the authors used ElliPro server and not another one? Is there any advantage of this tool compared to others? Answer: it is a tool that provides greater robustness to predictive models and has been shown to be a better server for conformational epitopes. - J Ponomarenko, H H Bui, W Li, N Fusseder, P E Bourne, A Sette, B Peters, ElliPro: a new structure-based tool for the prediction of antibody epitopes, BMC Bioinf. 9 (2008) 514. 2. Prediction of antigenic regions should be performed using different tools and individual analysis of accessibility, hydrophobicity, etc. And then compared the results to provide the “most probable” antigenic region. Answer: Given the robustness of the program, we do not consider the use of other tools. 3. The authors use the term “constitutional antigenic patches”. What does this mean? Is it a concept used by the bioinformatic tool? If so, they must explain what this is about. Answer: in the second version we will change the term to conformational epitopes for clarity. 4. Comparisons between aa sequences result in percentages of identity as a way to express the level of conservation or homology. However, proteins like phospholipases have variable molecular weights, which means that they aren’t similar in the whole length of their sequences. The authors must make clear that a portion of each protein is used to make the statements related to the identities and that the analysis is restricted to these areas. Answer: the PRALINE server makes an adjustment according to the length, based on the coverage between the proteins. The areas that are being conserved will be shown in the images. 5. Consequently, was the homology model made for the whole Ves s 1, Sol I 1, C. quinquefasciatus and C. hentzi phospholipases as whole proteins or just a portion of them? Answer: Yes, the whole protein model was made. 6. In the methods section, which templates were used by SWISS MODEL to predict the 3D model? Answer: The template that SWISS MODEL uses to predict tertiary structures is based on other homologous proteins with known tertiary structure. The server shows which is the base protein that is being taken for the creation of the new protein. This proteins will be show in the second version. 7. How was the superimposition of 3D structures presented in figure 5 performed?. The approach should be indicated in the methods. Answer: it was done using the matchmaker tool of the Pymol program. 8. The discussion is too long given the presented results. Sometimes the information provided is repetitive. For example, the second paragraph of the discussion is essentially previously mentioned in the results section and can be eliminated. Answer: we consider it important to make a short summary of the results for a better understanding of the discussion. 9. The third paragraph in the discussion section refers to carbohydrate determinants, which are not even superficially explored in this study. Although the observation referred to CCD can be mentioned in this manuscript, is not necessary to discuss this issue too much since there are no analysis for this matter in this paper. Answer: we preserve the written information from CCD because the information was suggested by the editor and another reviewer. 9. A similar evaluation of all the information provided in the discussion should be done. It is difficult to understand what the authors are trying to argue and how this is closely related to the actual results. A shortened and more precise discussion could greatly improve the manuscript. Answer: thanks we will take it into account for the second version Minor comments: 1. English and grammar needs to be revised. Many sentences need to be re-written to better disclose the message that the authors are trying to say, for example: - Allergic immune response from hymenopteran allergens has been studied in detail due to a high incidence of sting reactions to these insects… - A closed relationship among phospholipase allergens as shown, formed four nodes with a high phylogenetic relationship among them… - A better use of “comas” and “periods” must be done in order to improve the writing as well. For example: Molecular, structural, and immunological characterization of hymenopteran venom allergens is advanced, in total 75 allergens from 31 different species have been explored, and since phospholipases are a family of allergens with clinical and biological relevance, some proteins belonging to this order such as hyaluronidase and antigen V are also considered relevant to sensitization to this allergenic. Answer: English grammar is reviewed, as well as the use of points and commas. 2. In figure 3, what does “identities residues” mean? Answer: They could also be called conserved residues, these correspond to the amino acids that are being recognized as causing cross-reactivity, showing high identities between the different sequences analyzed. 3. If a total of 704 residues were identified and conserved among the phospholipases, why is figure 3 showing around 330 only? Answer: the PRALINE tool used for the alignment shows the number of residues taken into account for the analysis, what was done was to calculate how many residues of that total were conserved in those analyzed sequences. Thus, in figure 3, 934 of which 704 were conserved were taken into account in the analysis, this figure corresponds to 79% of the total residues. 4. We built four 3D models of the 18 phospholipases Ves s 1, Sol i 1, Culex quinquefasciatus and Centruroides hentzi… are you referring to “18” or “4”proteins? Answer: we will refer only to the 4 proteins that are named. They are those that failed to overlap due to the absence of structural homology. 5. Figures 3, 4 and 5 only show the multiple sequence alignments, but not the identified epitopes as the authors are pointing out. Please correct. Answer: we will select the linear epitope within the figure. 6. In group B (Bom p 1, Bom t 1, Api m 1, Api c 1) two analyses were conducted, the first with the presence of the Api c 1 allergen where a degree of identity of (35%) was found and the second without the allergen… why are you using parenthesis? Answer: It is fixed for the second version." } ] } ]	1	https://f1000research.com/articles/10-2
https://f1000research.com/articles/10-250/v1	29 Mar 21	{ "type": "Research Article", "title": "A new method for early detection of latent infection by ‘Candidatus Liberibacter asiaticus’ in citrus trees", "authors": [ "Kazuki Fujiwara", "Kenta Tomimura", "Toru Iwanami", "Takashi Fujikawa", "Kazuki Fujiwara", "Kenta Tomimura", "Toru Iwanami" ], "abstract": "Background: ‘Candidatus Liberibacter asiaticus’ (CLas) is a major causal agent of citrus greening disease. The disease primarily involves an asymptomatic, often latent infection of CLas. However, there is no effective technique to distinguish latent-infected trees from healthy ones. This study describes the development of a new detection method for latent CLas infection using cuttings. Methods: Root tissues regenerated from cuttings using symptomatic and asymptomatic citrus trees were prepared for real-time a polymerase chain reaction (PCR) test which was used to investigate latent CLas. When some of the regenerated roots were negative for CLas in the first real-time PCR assay, a subsequent cultivation in soils was performed using the CLas-negative cuttings. CLas development during cultivation was evaluated by a second real-time PCR assay using soil-grown roots from seedlings. Results: Previously, CLas had not been detected from leaves of the latent-infected trees in our greenhouse by real-time PCR. In this study, however, CLas was detected at a moderate frequency from the root tissues of cuttings derived from the latent-infected trees, by the same PCR test. For cuttings with regenerated roots that tested negative for CLas by real-time PCR, CLas was frequently detected from roots grown in nursery soil with autoclaving, after cultivation for a month or more. Conclusions: Latent infection with CLas was detectable by real-time PCR using root tissues regenerated by cuttings and roots grown in nursery soil with autoclaving. These results suggest that the new method of investigation would provide great opportunities for early detection of CLas in asymptomatic citrus trees from field surveys, and would accelerate the eradication practice of citrus greening.", "keywords": [ "Citrus greening disease", "cuttings", "latent infection", "real-time PCR" ], "content": "Introduction\n\nCitrus greening disease (huanglongbing; HLB) is one of the most devastating diseases for the citrus industry worldwide and is caused by the phloem-limited phytopathogenic bacteria ‘Candidatus Liberibacter’ spp. (Bové, 2006; Jagoueix et al., 1994). In particular, ‘Candidatus Liberibacter asiaticus’ (CLas) has been an important causal agent in many countries including parts of Asia (Bové, 2006). In Japan, the occurrence of citrus greening disease has been limited to areas of the south including the Okinawa prefecture and Oshima Islands, with Tokunoshima Island being the upper limit (Moji Plant Protection Station, 2012). Disease management has been carried out to prevent a further spread of the disease to citrus production areas on the main islands. A combination of PCR techniques for CLas detection and the disposal of infected citrus trees is the most practical approach to control the disease as there is currently no cure. On Kikaijima Island, located among the Amami Islands, a program for eradication of citrus greening disease was conducted in 2005 and had accomplished the eradication of the disease by 2012, resulting in an official declaration of eradication on Kikaijima Island (Moji Plant Protection Station, 2012). Currently, the eradication program is in progress on the larger Island of Tokunoshima, which is located to the south of Kikaijima Island.\n\nCitrus greening disease is not often distinguishable from physiological disorders. Early symptoms of citrus greening disease appear in the leaves and include yellowish, blotchy chlorosis and mottling of leaves (Bové, 2006). On the other hand, these symptoms resemble other disorders including virus infections and nutrient deficiencies (Tian et al., 2014). Also, the disease primarily involves latent infection of CLas. In this asymptomatic condition, CLas is often undetectable with PCR techniques because the pathogen has infected the asymptomatic tree at a very low density (Gottwald et al., 2007). Asymptomatic trees that are overlooked in the fields are an important source of infection through vector-mediated transmission by the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae) (Pelz-Stelinski et al., 2010). Because current disease management only allows us to identify citrus greening disease when CLas is detected by PCR and real-time PCR, the sensitivity and robustness of PCR detection is indispensable (Arredondo Valdés et al., 2016; Folimonova & Achor, 2010; Fujikawa & Iwanami, 2012). Recent studies have made significant progress in understanding the primary localization of CLas in roots before systemic distribution (Louzada et al., 2016; Park et al., 2018). CLas density in roots is relatively higher than that in leaves (Johnson et al., 2014). This suggests that root samples would be an alternative target for early detection of CLas. On the other hand, one drawback to using crude roots for detecting CLas would be the difficulties in processing DNA-based techniques. In contrast to leaf samples, root samples are not easily available in field surveys because of the hardened tissues in mature citrus trees, and it may be a struggle to dig up root tissues for further assays. In this study, we developed a new approach for early detection of CLas using root samples recovered from cuttings of CLas-infected trees. This approach allowed us to detect CLas from asymptomatic citrus trees that had previously tested negative when using leaves for conventional PCR and real-time PCR.\n\n\nMethods\n\nOne-year-old seedlings of rough lemon (Citrus jambhiri Lush.) were prepared in 12 cm-diameter × 24 cm-deep pots. Graft-inoculation of 12 seedlings was carried out with a small amount of bark tissue of CLas-infected flat lemon trees (Citrus depressa Hayata.), which has been maintained in a quarantined greenhouse under a special permission (26Y1214) issued by the Ministry of Agriculture, Forestry and Fisheries (Fujikawa et al., 2013). CLas infection on seven rough lemon trees was confirmed by both the development of foliar symptoms and the presence of CLas by real-time PCR using HLBas/HLBr/HLBp primer–probe set (Li et al., 2006). Five rough lemon trees did not develop any foliar symptoms, and CLas was undetectable from leaves over two years in a monthly survey with both the conventional PCR (Fujikawa et al., 2012) and real-time PCR using HLBas/HLBr/HLBp primer–probe set (Li et al., 2006). These inoculated but asymptomatic rough lemon trees (five pots in all) were used as asymptomatic trees for this study.\n\nCuttings were prepared from the upper part of young green shoots and branches, which were taken from seven symptomatic and five asymptomatic rough lemon trees. Cuttings of approximately 4 cm in length containing one leaf with no disease symptoms were selected and treated with 1% “Menedael” Fe (II) aq. fertilizer (Menedael Co., Ltd., Osaka, Japan; Cat. No. 242461) and then dipped in “Rooton”, a synthetic auxin α-naphthyl acetamide, (Sumitomo chemical garden products inc., Tokyo, Japan; JAN code 4975292090625) for promotion of rooting. The cuttings that had been treated were planted in a box that contained autoclaved “Kanuma” pumice and kept at approximately 32°C for a month under natural light/dark conditions, covered with a plastic film. Care was taken to keep the Kanuma pumice gently wet but not saturated to ensure optimum rooting. Roots that regenerated a month after cutting were used for CLas detection in the real-time PCR (Li et al., 2006).\n\nFor detecting CLas in cuttings with root-sprouts that tested negative in the first PCR test, subsequent cultivation of the cuttings in various soils was carried out in a quarantined greenhouse and monitored for two months. Five cuttings were individually transplanted into the following soils: two commercial soils which were “Kenbyo” nursery soil (Yaenougei Co., Ltd., Nagasaki, Japan) and “Sanyo composted bark” soil (Sanyo Chip Co., Ltd., Yamaguchi, Japan); one natural soil from an area where citrus greening disease has occurred, namely “Kunigami Maaji” clay soil collected from Kunigami-son village (around N26°44'44.8\", E128°10'42.0\"), Okinawa Prefecture; and two natural soils from areas where citrus greening disease has not occurred, which were Acrisols collected from Isen-cho (around N27°40'25.2\", E128°56'16.2\"), Tokunoshima Islands, Kagoshima Prefecture and Andosols collected from Tarumizu-city (around N31°28'38.1\", E130°43'19.5\"), Kagoshima Prefecture. The natural soils were collected in 2016. During soil sampling, the surface litter and soil at the sampling spot was removed using a spade. Using a plow at each sampling site, each natural soil was collected up to a depth of 10 cm and bulked. The precise locations of the sampling sites are not disclosed due to landowner privacy reasons. All soils were prepared with and without autoclaving. The autoclaving process was repeated twice. During the cultivation, 0.1g of “Hyponex” powder fertilizer (HYPONeX Japan Co., Ltd., Tokyo, Japan; JAN code 4977517003052) and one tablet of Aid-ball Ca (Sumitomo chemical garden products Inc., Tokyo, Japan; JAN code 4975292110118) were applied once a month. A single leaf and root were collected from each cutting on a monthly basis to determine the CLas infection status using real-time PCR (Li et al., 2006).\n\nLeaf samples were cleaned and wiped with a kimwipe paper (Kimberly Clark Corp., Irving, TX) impregnated with 70% ethanol. Midribs were excised and chopped into 2–3 mm pieces, providing approximately 0.05 (±0.01 SD) g of the midribs for DNA extraction. For the root samples, a single root was vortexed vigorously to remove soils. Approximately 4 cm of the root segment was cut out from the bottom of the root and then wiped with a kimwipe impregnated with 70% ethanol. All sample materials were individually ground with a mortar and pestle, and then subjected to DNA extraction using the commercial kit DNeasy Plant Mini Kit (QIAGEN, Valencia, CA; Cat. No. 69106) according to the manufacturer’s instructions. DNA was eluted in 200 μL of a given AE buffer. CLas detection in cuttings was performed by TaqMan probe real-time PCR in a QuantStudio 5 real-time PCR system (Thermo Fisher Scientific Inc., Waltham, MA) using the HLBas (5’-TCGAGCGCGTATGCAATACG-3’) / HLBr (5’-GCGTTATCCCGTAGAAAAAGGTAG-3’) / HLBp (6-FAM-AGACGGGTGAGTAACGCG-BHQ-1) primer–probe set (Li et al., 2006). Premix Ex Taq (Probe qPCR) (Takara Bio Inc., Shiga, Japan) was used according to the manufacturer’s protocol. The PCR reaction volume of 20 µL contained 0.4 µL of each primer, 10 µL of Premix Ex Taq, 0.8 µL of HLBp, 0.4 µL of ROX Reference Dye II, 2 µL DNA template, and 6 µL of sterile distilled water. The real-time PCR consisted of pre-denaturation at 95°C for 30 s, and 45 cycles of denaturation at 95°C for 15 s and annealing/extension at 60°C for 1 min. CLas concentration was calculated based on the relationship between quantity and molarity of CLas 16S rDNA (as outlined by Degen et al., 2006), and the standard curve analysis was determined using the amount of template DNA adjusted to the DNA content in 108 cells/μL and a 10-fold dilution series. The CLas density in leaves was calculated from the total amount of DNA eluted from leaves according to the representative standard curve (y = −1.538x + 42.278, R2 = 0.9999). The results of the CLas development were analyzed using Tukey-Kramer multiple comparison test in the R statistical software (v. 3.5.0)\n\nDifferences were considered significant when P < 0.01.\n\n\nResults\n\nMost of the cuttings from asymptomatic trees survived and were well rooted (Figure 1a–c), but the regeneration of cuttings using symptomatic trees was evidently influenced by CLas infection. A total of 224 clones regenerated from 240 prepared cuttings using asymptomatic trees whereas a total of 24 clones were recovered from 45 prepared cuttings using symptomatic trees. One root sample was collected from each cutting and these were used for the primary PCR test. CLas was detected in a total of 115 cuttings in the real-time PCR (Fujiwara, 2021a), which corresponds to a 51.3% detection rate (Table 1), while none of the cuttings showed foliar symptoms of citrus greening. In addition to this test, nine leaf samples from cuttings showing CLas negative in roots were randomly selected and subjected to real-time PCR, the results of which showed no detection of CLas within the leaf samples. In regeneration of cuttings using symptomatic trees, CLas was detected in all root samples, which corresponds to a 100% detection rate (Table 1), as was detected in the symptomatic leaves.\n\nPreparation of cuttings using asymptomatic trees; a, cuttings grown in Kanuma soil a month after preparation; b–c, seedlings recovered from cuttings showing newly developed roots.\n\n* Roots re-generated a month after cutting were used to detect CLas by the real-time polymerase chain reaction. Raw data is provided on Figshare (Fujiwara, 2021a).\n\nA subsequent cultivation test was performed (Fujiwara, 2021b) to assess the CLas recovery from latent infection using the cuttings which showed no CLas detection in the primary PCR test (Supplementary Figure 1, Extended data (Fujiwara, 2020)). To examine suitable soil types for CLas development in cultivation, we tested five different types of soils including two commercial soils (“Kenbyo” nursery soil and “Sanyo composted bark” soil) and three natural soils (Acrisols, Andosols, and “Kunigami Maaji” soil). Overall, a cultivation of the cuttings in prepared soils successfully provided CLas detection by real-time PCR from asymptomatic trees (Figure 2). In the nursery soil, CLas was detectable from both leaves and roots a month after transplanting (P < 0.01). The density of CLas in seedlings grown in the “Kenbyo” nursery soil with autoclaving (3.4 × 105 cells/leaf and 2.1 × 105 cells/root) was relatively higher than that in the same soil without autoclaving (3.8 × 103 cells/leaf and 4.9 × 103 cells/root), and the detection was stable throughout the cultivation for two months (Figure 2; Supplementary Figure 2, Extended data (Fujiwara, 2020)). In contrast, although CLas was likely to be detected in both leaves and roots grown in a composted soil (1.7 × 103 cells/leaf and 4.5 × 101 cells/root with autoclaving), the CLas density detected in cultivations in the composted soil without autoclaving fluctuated highly (Figure 2; Supplementary Figure 3, Extended data (Fujiwara, 2020)). Seedlings grown in composted soil without autoclaving showed CLas development a month after cultivation, no CLas was detected in the subsequent cultivation. In the natural soils, which comprised Acrisols, Andosols, and “Kunigami Maaji” soils, it was also the case that CLas detection fluctuated in both the leaves and roots similar to that in the composted bark soil without autoclaving (Figure 2; Supplementary Figure 4–6, Extended data (Fujiwara, 2020)). Autoclaving of the Andosols and “Kunigami Maaji” soil had little effect on the propagation of CLas in the cuttings, showing 2.3 × 103 cells/leaf and 1.1 × 104 cells/root in Andosols without autoclaving; 5.2 × 102 cells/leaf and 9.5 × 102 cells/root in a Kunigami Maaji soil without autoclaving. In Acrisols, CLas development was relatively varied compared with other natural soils and appeared to be affected by the soil conditions with autoclaving (5.4 × 103 cells/leaf and 2.2 × 104 cells/root with autoclaving) (Figure 2; Supplementary Figure 4, Extended data (Fujiwara, 2020)). In short, the soil conditions of a nursery soil treated with autoclaving was suitable for early detections of CLas recovered from latent infection using cuttings.\n\nAverage of CLas density two months after cultivation was determined by real-time PCR and calculated using CLas density in five individual seedlings shown in Supplemental Figures 2–6 (see Extended data). CLas recovered from latent infection was likely to be detected from leaves and roots using nursery soil with autoclaving. Statistical significance was shown (*P < 0.01, Tukey-Kramer multiple comparison test). +: with autoclaved, -: without autoclaved.\n\n\nDiscussion\n\nThis study showed that while it remains difficult to identify CLas from latent infection in host trees, latently infected CLas can be detectable by real-time PCR using roots regenerated from cuttings and grown in autoclaved nursery soil. The results indicated that detection from cuttings with or without cultivation in autoclaved nursery soil can be practically applicable to detect CLas from latently infected trees. In field tests, it might be possible to detect CLas directly from the root samples of latently infected trees, but in some seasons and locations digging on site and finding new, fine, roots would be extremely difficult. Instead, the CLas detection from cuttings reported here is suitable in any surrounding as long as twigs are available.\n\nSoil chemicals and microorganisms evidently affect the occurrence and distribution of CLas in citrus (Blaustein et al., 2017) as is the case with other soil-borne diseases (Philippot et al., 2013). CLas is likely to accumulate in roots prior to systemic distribution in citrus trees (Johnson et al., 2014) and requires CLas-associated microbiota in the host plant for its own survival (Fujiwara et al., 2018). This suggests that disturbance of CLas-associated microbiota by soil microorganisms may affect CLas development in citrus trees. A subset of CLas-associated microbiota in roots was structured by CLas infection, resulting in distinct microbial and metabiological profiles in roots (Padhi et al., 2019; Zhang et al., 2017). Thus, soil and rhizosphere management may be critical for disease control management. On the other hand, there is a concern that improved root integrity slows CLas growth and delays disease development, making it difficult to detect CLas in roots and other plant tissues. Our study has shown that CLas recovery from latent infection varied with soil type and conditions. While we still need to clarify the interplay between soil functions and CLas behaviors, further accumulation of knowledge on the relationship between root zone and the occurrence of citrus greening diseases is expected to enable more effective monitoring and detection of CLas in roots (Blaustein et al., 2018).\n\nA citrus greening disease eradication program in Japan has been promoted by both the early detection of CLas from leaf samples using PCR methods and the removal of the infected trees. Detection of CLas from latent infection was once carried out by a validation method using grafting over six months (Figure 3a). The disease management has been successful in increasing citrus greening disease-free areas in the Okinawa prefecture and Amami Islands. In addition to the conventional survey, a new approach developed in this study could be provided as part of an eradication program for finding latent infection of CLas and is expected to contribute to time-and cost-efficient validation of CLas infection detection. In practice, the process of a new approach consists of two sequential steps (Figure 3b; Supplementary Figure 7a-b, Extended data (Fujiwara, 2020)). As foliar samples from an asymptomatic tree test negative in the primary PCR test, cuttings should be prepared from the asymptomatic tree. Roots regenerated from the cuttings are then used for CLas detection in the second PCR test (Figure 3b; Supplementary Figure 7a, Extended data (Fujiwara, 2020)). If CLas is detected from any of the cuttings, the asymptomatic tree would be removed from the field. Next, a subsequent cultivation test is conducted as the third step if all cuttings from the asymptomatic tree test negative in the second PCR test (Figure 3b; Supplementary Figure 7b, Extended data (Fujiwara, 2020)). Root samples are collected within a few months after cultivation, and the results would allow us to decide whether or not to eliminate the asymptomatic tree. When CLas infection is not validated throughout the process, further monitoring or a re-start of the survey would be followed if necessary.\n\nA new scheme for investigating latent infection of Candidatus Liberibacter asiaticus (CLas). Conventional (a) and new (b) approaches for validation of CLas infection in asymptomatic trees.\n\nThe techniques described in this paper are only applicable for citrus cultivars that root readily by cutting. These cultivars include lemon, trifoliate orange, murcot, Cleopatra mandarin, as well as the flat lemon that is common in the citrus greening-affected subtropical islands of Japan. For satsuma mandarin, oranges, and others that root poorly by cutting, propagation by air-layering would be needed. Exploration on media considering plant hormone formula and temperature conditions for promoting rooting from these cultivars is in progress in our laboratory.\n\n\nConclusion\n\nThe latent infection of CLas is detectable with the new detection method using cuttings. Our study revealed that the autoclaved nursery soils allowed for early detection of CLas. The new method using cuttings is time efficient in CLas surveying, in contrast to the conventional method which uses grafting. It is expected that the new method may provide opportunities for identification and validation of other ‘Candidatus Liberibacter’ spp. under quarantine conditions, as well as providing opportunities in monitoring surveys of citrus greening disease.\n\n\nData availability\n\nFigshare: Raw data for “Detection of CLas from roots recovered from cuttings” shown in Table 1. https://doi.org/10.6084/m9.figshare.13985021.v1 (Fujiwara, 2021a).\n\nThis project contains the following underlying data:\n\n- Raw Data 1 (raw, unaveraged real-time PCR data).\n\nFigshare: Raw data for “A subsequent cultivation in different soil types using CLas-negative cuttings” shown in Supplementary Figures 2–6. https://doi.org/10.6084/m9.figshare.13985198.v1 (Fujiwara, 2021b).\n\nThis project contains the following underlying data:\n\n- Raw Data 2 (raw, unaveraged real-time PCR data).\n\nFigshare: Supplementary Figures 1–7. https://doi.org/10.6084/m9.figshare.13466676.v1 (Fujiwara, 2020).\n\nThis project contains the following extended data:\n\n- Figure 1. Schematic representation of the experimental design.\n\n- Figure 2. A subsequent cultivation test for CLas detection in “Kenbyo” nursery soil using seedlings with no CLas detection in a primary PCR test.\n\n- Figure 3. A subsequent cultivation test for CLas detection in “Sanyo composted bark” soil using seedlings with no CLas detection in a primary PCR test.\n\n- Figure 4. A subsequent cultivation test for CLas detection in Acrisols using seedlings with no CLas detection in a primary PCR test.\n\n- Figure 5. A subsequent cultivation test for CLas detection in Andosols using seedlings with no CLas detection in a primary PCR test.\n\n- Figure 6. A subsequent cultivation test for CLas detection in “Kunigami Maaji” soil using seedlings with no CLas detection in a primary PCR test.\n\n- Figure 7. A new approach for validation of CLas infection in asymptomatic trees.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nEthics\n\nNo special permissions or quarantines were required for this study.", "appendix": "Acknowledgements\n\nWe thank Yuko Shimizu from Okinawa Prefectural Plant Protection Center. We also thank Katsuhiko Miyaji, Yoshihiro Ogawa, and Tomohiro Fukumoto from Kagoshima Prefectural Institute for Agricultural Development for supporting the soil collection. We are also grateful to Mariko Taguchi, Akane Sasaki, Hiroe Hatomi, and Yuko Fujii from NARO for their support throughout the experiments.\n\n\nReferences\n\nArredondo Valdés R, Ortiz JCD, Beache MB, et al.: A review of techniques for detecting Huanglongbing (greening) in citrus. Can J Microbiol. 2016; 62(10): 803–811. PubMed Abstract \| Publisher Full Text\n\nBlaustein RA, Lorca GL, Meyer LJ, et al.: Defining the core citrus leaf- and root-associated microbiota: Factors associated with community structure and implications for managing huanglongbing (citrus greening) disease. Appl Environ Microbiol. 2017; 83(11): e00210–17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlaustein RA, Lorca GL, Teplitski M: Challenges for Managing Candidatus Liberibacter spp. (Huanglongbing Disease Pathogen): Current Control Measures and Future Directions. Phytopathology. 2018; 108(4): 424–435. PubMed Abstract \| Publisher Full Text\n\nBové JM: Huanglongbing: a destructive, newly emerging, century-old disease of citrus. J Plant Pathol. 2006; 88(1): 7–37. Reference Source\n\nDegen HJ, Deufel A, Eisel D, et al.: PCR Application Manual. 3rd Ed. (Roche Diagnostics GmbH, Mannheim). 2006. Reference Source\n\nFolimonova SY, Achor DS: Early events of citrus greening (huanglongbing) disease development at the ultrastructural level. Phytopathology. 2010; 100(9): 949–958. PubMed Abstract \| Publisher Full Text\n\nFujikawa T, Iwanami T: Sensitive and robust detection of citrus greening (huanglongbing) bacterium “Candidatus Liberibacter asiaticus” by DNA amplification with new 16S rDNA-specific primers. Mol Cell Probes. 2012; 26(5): 194–197. PubMed Abstract \| Publisher Full Text\n\nFujikawa T, Miyata SI, Iwanami T: Convenient Detection of the Citrus Greening (Huanglongbing) Bacterium ‘Candidatus Liberibacter asiaticus’ by Direct PCR from the Midrib Extract. PLoS One. 2013; 8(2): e57011. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFujiwara K: Raw data for \"Detection of CLas from roots recovered from cuttings\" shown in Table 1. figshare. Dataset. 2021a. http://www.doi.org/10.6084/m9.figshare.13985021.v1\n\nFujiwara K: Raw data for \"a subsequent cultivation in different soil types using CLas-negative cuttings\" shown in Supplementary Figures 2-6. figshare. Dataset. 2021b. http://www.doi.org/10.6084/m9.figshare.13985198.v1\n\nFujiwara K: Supplementary Figures 1-7. figshare. Figure. 2020. http://www.doi.org/10.6084/m9.figshare.13466676.v1\n\nFujiwara K, Iwanami T, Fujikawa T: Alterations of Candidatus Liberibacter asiaticus-Associated Microbiota Decrease Survival of Ca. L. asiaticus in in vitro Assays. Front Microbiol. 2018; 9: 3089. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGottwald TR, da Graça JV, Bassanezi RB: Citrus Huanglongbing: The pathogen and its impact. Plant Health Prog. 2007; 8(1). Publisher Full Text\n\nJagoueix S, Bove JM, Garnier M: The phloem-limited bacterium of greening disease of citrus is a member of the alpha subdivision of the Proteobacteria. Int J Syst Bacteriol. 1994; 44(3): 379–386. PubMed Abstract \| Publisher Full Text\n\nJohnson EG, Wu J, Bright DB, et al.: Association of ‘Candidatus Liberibacter asiaticus’ root infection, but not phloem plugging with root loss on huanglongbing-affected trees prior to appearance of foliar symptoms. Plant Pathology. 2014; 63(2): 290–298. Publisher Full Text\n\nLi W, Hartung JS, Levy L: Quantitative real-time PCR for detection and identification of Candidatus Liberibacter species associated with citrus huanglongbing. J Microbiol Methods. 2006; 66(1): 104–115. PubMed Abstract \| Publisher Full Text\n\nLouzada ES, Vazquez OE, Braswell WE, et al.: Distribution of 'Candidatus Liberibacter asiaticus' Above and Below Ground in Texas Citrus. Phytopathology. 2016; 106(7): 702–709. PubMed Abstract \| Publisher Full Text\n\nMoji Plant protection station: Emergency action and eradication on citrus greening disease in Kikai Island. Plant Protection. (In Japanese). 2012; 66(6): 348–349. Reference Source\n\nPadhi EMT, Maharaj N, Lin SY, et al.: Metabolome and Microbiome Signatures in the Roots of Citrus Affected by Huanglongbing. Phytopathology. 2019; 109(12): 2022–2032. PubMed Abstract \| Publisher Full Text\n\nPark JW, Louzada ES, Evan Braswell W, et al.: A new diagnostic real-time PCR method for huanglongbing detection in citrus root tissue. J Gen Plant Pathol. 2018; 84(5): 359–367. Publisher Full Text\n\nPelz-Stelinski KS, Brlansky RH, Ebert TA, et al.: Transmission Parameters for Candidatus Liberibacter asiaticus by Asian Citrus Psyllid (Hemiptera: Psyllidae). J Econ Entomol. 2010; 103(5): 1531–1541. PubMed Abstract \| Publisher Full Text\n\nPhilippot L, Raaijmakers JM, Lemanceau P, et al.: Going back to the roots: the microbial ecology of the rhizosphere. Nat Rev Microbiol. 2013; 11(11): 789–799. PubMed Abstract \| Publisher Full Text\n\nTian S, Lu L, Labavitch JM, et al.: Spatial imaging of Zn and other elements in Huanglongbing-affected grapefruit by synchrotron-based micro X-ray fluorescence investigation. J Exp Bot. 2014; 65(4): 953–964. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang Y, Xu J, Riera N, et al.: Huanglongbing impairs the rhizosphere-to-rhizoplane enrichment process of the citrus root-associated microbiome. Microbiome. 2017; 5(1): 97. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "82413", "date": "21 Apr 2021", "name": "Siti Subandiyah", "expertise": [ "Reviewer Expertise Plant Pathology", "some on the similar area studying of huanglongbing" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript is well written with good English throughout the whole paper. However, a little improvement is needed in the Methods (e.g. quantification of the inoculum source) or when some methodological sentences may need to be moved from the Results to Methods.\nThe Discussion may be improved on the explanation of the soil characteristic deferences among the soil types used in the experiment. The cultivar(s) recommended for root stocks when air layering propagation may be needed.\nI have provided some further notes here.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "82414", "date": "29 Apr 2021", "name": "Madhurababu Kunta", "expertise": [ "Reviewer Expertise Citrus Pathology", "Citrus Pathogen Detection", "Horticulture" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study contributes to detection of CLas from asymptomatic trees using root samples which is highly useful for the stakeholders to take appropriate HLB management strategies and to prevent the spread of the disease. This is a valuable scientific report that provides early and pre-symptomatic Candidatus L asiaticus (CLas) detection in citrus using root samples. However, the authors of the manuscript used HLBaspr 16 S rDNA-based qPCR test to detect CLas, causal organism for citrus HLB, from the citrus roots. I recommend that the authors test for CLas all the samples using another primer set targeting a region other than 16S rDNA (for example, RNR) to confirm the results. I would be glad to review the manuscript if the authors can conduct the tests and submit a revised manuscript based on my recommendations. This recommendation is based on the previous scientific reports.\nKunta et al. (2014)1 tested both leaf and root samples and concluded that HLBaspr primers are not suitable for CLas detection in root samples due to false positives. The reason to include my paper is that it was the first study published in 2014 that determined that HLBaspr based qPCR to amplify 16S rDNA region is not suitable to detect CLas from citrus root samples due to non-specific amplification from non-target organisms which leads to false positive results. However, HLBaspr works excellent for Clas detection in the leaf samples.\nAnother study, Shin et al. (2018)2, reported that Bradyrhizobium sp. closely related CLas within Rhizobiales contributed to the false postives in the roots when HLBaspr primers targeting 16rDNA are used. This paper was published in 2018, which describes that rhizobiales such as Bradyrhizobium (Bradyrhizobium 16S rDNA sequences are highly similar to CLas) could interfere and lead to unreliable CLas detection in roots. This study also recommends that primers other than HLBaspr should be used to avoid potential cross reactions with members of Rhizobiales for accurate CLas detection.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-250
https://f1000research.com/articles/10-248/v1	29 Mar 21	{ "type": "Study Protocol", "title": "Does the speed of sternal retraction during coronary artery bypass graft surgery affect postoperative pain outcomes? A randomized controlled trial protocol", "authors": [ "Dimitri Petsikas", "Craig Stewart", "Rachel Phelan", "Rene Allard", "Michael Cummings", "Deborah DuMerton", "Joel Parlow", "Darrin Payne", "Robert Tanzola", "Louie Wang", "Tarit Saha", "Dimitri Petsikas", "Craig Stewart", "Rachel Phelan", "Rene Allard", "Michael Cummings", "Deborah DuMerton", "Joel Parlow", "Darrin Payne", "Robert Tanzola", "Louie Wang" ], "abstract": "Background: Chronic pain is a serious health issue impacting both the quality of life and productivity of patients. Chronic post-sternotomy pain (CPSP) is characterized by numbness, severe tenderness on palpation, allodynia, as well as constant pain across the anterior chest wall that can persist for months to years after sternotomy. All patients experience early post-operative pain following coronary artery bypass graft (CABG); unfortunately, approximately 30-40% of CABG patients subsequently develop CPSP.\n\nMethods: The current study is a prospective, double-blinded, randomized controlled trial. A sample size of 316 randomly assigned patients (n=158 per group) will provide an 80% power at a 2-sided α of 0.05 to detect a 40% decrease in CPSP incidence at 6 months. Eligible patients scheduled for elective, primary coronary artery bypass graft surgery will be randomly assigned to the CONTROL group, in which sternal retraction is conducted over 30 seconds (as per standard practice); or the SLOW group, in which sternal retraction is achieved over 15 minutes. Surgical and perioperative anesthesia protocols between the two groups are otherwise the same. Our primary outcome is the incidence of CPSP at 6 months. Secondary outcomes are: CPSP incidence at 3 and 12 months, daily sternal incision pain intensity (numeric rating scale (NRS)) at rest and while coughing, and daily analgesic consumption while in hospital and at 7 days postoperatively; pain quality, quality of life, and pain interference with daily function at 3, 6, and 12 months post-operatively.\nDiscussion: Our randomized controlled trial will determine whether retracting the sternum more slowly for exposure of the heart during CABG surgery will decrease the incidence and/or severity of CPSP.\nClinicalTrials.gov registration: NCT02697812 (03/03/2016", "keywords": [ "Coronary Artery Bypass Surgery", "chronic pain" ], "content": "Abbreviations\n\nChronic Post-Sternotomy Pain (CPSP)\n\nCoronary Artery Bypass Graft (CABG)\n\nInternal Mammary Artery (IMA)\n\nActivated Clotting Time (ACT)\n\nBasic History Questionnaire (BHQ)\n\nBrief Pain Inventory-short form (BPI-SF)\n\n36-Item Short Form Health Survey (SF-36)\n\nShort form-McGill pain questionnaire (SF-MPQ)\n\nDouleur Neuropathique 4 (DN4)\n\nNumeric Rating Scale (NRS)\n\nResearch Ethics Board (REB)\n\n\nIntroduction\n\nCoronary artery bypass surgery and chronic post-sternotomy pain. All patients experience pain following coronary artery bypass graft (CABG) surgery both within the early and intermediate postoperative periods. Severe pain may compromise the patients’ recovery by affecting breathing, mobility, and sleep. Inadequately controlled postoperative pain has been associated with increased cardiovascular complications, pneumonia, hyper-coagulability, delirium and wound infection1. Of the patients readmitted following CABG, many report recurrent ischemic cardiac chest pain while others suffer from non-cardiac chest pain. Pain is the fifth most common reason for readmission within 4 weeks of CABG2. Severe, inadequately controlled postoperative pain may also be a predisposing factor for developing chronic post-sternotomy pain (CPSP)3. Although CPSP is now becoming recognized as a serious health issue with a major impact on quality of life and productivity, this complication has traditionally been overlooked and/or dismissed as an unavoidable consequence of surgery. CPSP is characterized by numbness, severe tenderness on palpation, allodynia (pain response to normally non-painful stimuli), as well as constant pain across the anterior chest wall4, and this pain can persist for up to 16 months5. Inter-scapular and shoulder girdle pain may also be present.\n\nEstimates of the incidence of CPSP vary widely depending upon the population, the pain measures used and the time-point under consideration. Although the prevalence of CPSP has been reported as high as 52% 6 months following CABG6, most report an incidence of approximately 30–40% between 6 and 16 months postoperatively5–7. Given that almost 500,000 CABG procedures were performed within North America during 20098,9, this translates to a significant number of patients adversely affected by CPSP. Despite the major impact of chronic post-surgical pain on quality of life, productivity and associated medical expenses, it is still an area of research that receives relatively little attention10 and the actual process by which acute postsurgical pain transitions into chronic pain receives even less attention11.\n\nThe etiology of CPSP is complex and not well understood. As mentioned previously, the development of CPSP is positively correlated with severe and inadequately controlled acute post-operative pain. However, the fact that not all who experience severe post-operative pain go on to develop CPSP, suggests that other factors such as the genetic variation in the underlying pain mechanisms and/or prior experiences may alter one’s response to painful stimuli both in the short and long term. It has also been suggested that techniques associated with internal mammary artery (IMA) harvesting increase the likelihood of CPSP12,13 but even patients who do not undergo IMA harvesting may report an equally high incidence of CPSP14. The high incidence of CPSP associated with cardiac surgery can likely be attributed largely to the extensive nerve and tissue damage15 from the shear forces applied during rapid sternal retraction. Shear forces are not only applied in the lateral-dorsal-ventral directions but also on the rostral-caudal plane.\n\nTo our knowledge, no clinical investigations have examined the sternal retraction maneuver as a causal determinant in the severity of postoperative pain or the subsequent development of CPSP. However, Bolotin et al. (2007)16 did use anesthetized sheep to demonstrate that controlled retraction forces (and hence increased time) to sternal retraction, resulted in significantly lower maximal and average applied forces. In addition, using controlled force to allow equivalent exposure resulted in significantly reduced alterations in blood pressure and heart rate thought to be indicative of reduced pain and/or stress compared to standard sternal retraction forces. A similar study with lateral open thoracotomies in sheep with standard versus slower retraction, observed significant reductions in applied forces and this was associated with reduced animal stress and tissue damage17. Several other investigations have explored the use of mini-sternotomies for aortic valve surgery18–20 with mixed results on pain, length of hospital stay, blood loss and/or pulmonary function. The one study which compared mini vs. standard sternotomy for CABG examined only postoperative pulmonary function and observed no difference21.\n\nAcute post-sternotomy pain occurs as a result of tissue and nerve damage (intercostal nerves and thoracic nerves (T2-T6) which innervate the sternum and/or ribs) which provokes direct nociceptive input. However, with the physical trauma, numerous inflammatory mediators (i.e., bradykinin, substance P, histamine, 5-Hydroxytryptamine, adenosine triphosphate, nitric oxide, prostanoids and leukotrines) are released and these induce pain and inflammation at more remote sites22. Furthermore, these inflammatory mediators induce sensitization of both peripheral and central neural pathways. It is the sensitization of central pain-signalling neurons from intense and prolonged nociceptive activity which is thought to result in the transition from acute to chronic pain22–24. The prolonged sensory input provokes abnormal responses to low-threshold mechanoreceptor activity (allodynia and hyperalgesia) and central nervous system remodelling that contributes to this persistent pain state.\n\n\nObjectives\n\nThe overall objective of the current investigation is to determine whether increasing the time to achieve sternal retraction to 15 minutes (from the standard ~30 seconds), will result in reduced acute and chronic post-sternotomy pain and an improved quality of life. We hypothesize that increasing the time to sternal retraction will result in reduced required force, less physical trauma, less inflammation, and less nerve damage. Specifically, we expect SLOW steady sternal retraction (occurring over 15 minutes) to: 1) reduce the incidence of CPSP by 40% and 2) significantly improve quality of life 6 months postoperatively.\n\n\nMethods\n\nThis is a prospective, double-blind, randomized controlled superiority trial. It will take place at Kingston General Hospital site, Kingston Health Sciences Centre, Ontario, Canada which is a medium size (~ 470 beds), academic, tertiary care centre.\n\nInclusion criteria\n\nElective primary coronary artery bypass surgery\n\nUnderstanding and provision of written informed consent\n\nAge > 18 years\n\nExclusion criteria\n\nAny combined or redo cardiac procedure\n\nCurrent alcohol or substance abuse\n\nPre-existing chronic pain requiring chronic analgesic use\n\nRest pain in proposed surgical area at baseline, preoperatively\n\nChronic Steroid use\n\nInability to perform post-operative assessments\n\nPlanned bilateral internal thoracic artery harvest for bypass conduits\n\nRheumatoid arthritis\n\nPrior radiation to the chest\n\nCoronary artery bypass surgeries for participants in the study will be conducted by cardiac surgeon Dr. Dimitri Petsikas (MD, FRCSC) and colleagues at Kingston Health Sciences Centre.\n\nThe Queen’s University Health Sciences and Affiliated Teaching Hospitals Research Ethics Board approved this study (ANAE-233-13, May 21, 2013). Written, informed consent for participation will be obtained from all participants25.\n\n\nInterventions\n\nTo our knowledge, no clinical investigations have examined the sternal retraction maneuver as a causal determinant in the severity of postoperative pain or the subsequent development of CPSP. However, Bolotin et al. (2007)16 did use anesthetized sheep to demonstrate that controlled retraction forces (and hence increased time) to sternal retraction, resulted in significantly lower maximal and average applied forces. In addition, using controlled force to allow equivalent exposure resulted in significantly reduced alterations in blood pressure and heart rate thought to be indicative of reduced pain and/or stress compared to standard sternal retraction forces. A similar study with lateral open thoracotomies in sheep with standard versus slower retraction, observed significant reductions in applied forces and this was associated with reduced animal stress and tissue damage17. Several other investigations have explored the use of mini-sternotomies for aortic valve surgery18–20 with mixed results on pain, length of hospital stay, blood loss and/or pulmonary function. The one study which compared mini vs. standard sternotomy for CABG examined only postoperative pulmonary function and observed no difference21.\n\nThus, the overall objective of the current investigation is to determine whether increasing the time to achieve sternal retraction to 15 minutes (from the standard ~30 seconds), will result in reduced acute and chronic post-sternotomy pain and an improved quality of life.\n\nAnesthesia. Patients will receive general anesthesia with endotracheal intubation, using moderate dose opioid (fentanyl 10–20 mcg/kg or sufentanil 1–2 mcg/kg), inhalational anesthesia (isoflurane or sevoflurane up to 1.5 MAC), propofol, midazolam and succinylcholine and/or rocuronium. Monitoring will include standard Canadian Anesthesia Society (CAS) monitors, an invasive arterial line, a central venous line and a Foley catheter. In preparation for cardiopulmonary bypass, all patients will receive intravenous unfractionated heparin to achieve an activated clotting time (ACT) > 480 seconds. The ACT will be maintained at this level with additional heparin as necessary. Following separation from cardiopulmonary bypass, the heparin effects will be reversed with protamine administration to achieve an ACT within 10% of baseline. On admission to the intensive care unit, patients will remain sedated using a propofol infusion 25–75 mcg/kg/min until extubation; and patients will receive protamine 50 mg/hr for 3 consecutive hours.\n\nSurgery. Participants will then receive one of two surgical conditions:\n\nStandard “normal speed” sternal retraction (approximately 30 seconds)\n\nMedian sternotomy carried out, and sternal hemostasis obtained\n\nSternum retracted to a width which will allow adequate exposure to allow performance of the coronary bypass procedure at the individual surgeon’s discretion\n\nMaximum distance between sternal edges at full retraction will be measured with distance calipers\n\nThe sternal retractor will then be removed and the left or right internal thoracic artery will be harvested as a bypass conduit\n\nSternal retractor will be re-inserted and sternum retracted to the previously determined MAXIMAL STERNAL RETRACTION distance. The coronary bypass procedure will then proceed at the surgeon’s discretion, (i.e. number and type of bypass grafts)\n\nSlow sternal retraction (approximately 15 minutes)\n\nMedian sternotomy carried out and sternal hemostasis obtained\n\nSternum retracted to a width allowing adequate exposure to perform the coronary bypass procedure over 15 minutes\n\nMaximum distance between sternal edges at full retraction will be measured with distance calipers\n\nThe sternal retractor will then be removed and the left or right internal thoracic artery will be harvested as a bypass conduit\n\nSternal retractor will be re-inserted and sternum retracted to the previously determined MAXIMAL STERNAL RETRACTION distance. The coronary bypass procedure will then proceed at the surgeon’s discretion, (i.e. number and type of bypass grafts)\n\nAll cases will be performed via median sternotomy with cardiopulmonary bypass support, using standard aortic and single two-stage right atrial venous cannulation. Intermittent antegrade blood cardioplegia will be utilized. All bypasses will be performed under a single cross-clamp. All patients will have a single 32 French anterior mediastinal drain. A 28 French chest tube will be placed in either or both of the pleural spaces should they be opened at any point during the surgery. All chest drains will be placed to suction at -20 cmH2O.\n\nCriteria for discontinuing or modifying allocated interventions. At any time, either prior to anaesthetic induction or afterward, should the patient become electrocardiographically or hemodynamically unstable, he/she will be withdrawn from the intervention and the operation will proceed as per standard of care for optimal patient safety. Participants will be withdrawn from the study if there is a change in planned surgical procedure.\n\nStudy staff, who otherwise have no involvement in the study in terms of patient enrolment or assessment, will be in the operating room (OR) to facilitate protocol compliance with the intervention and measure the time to achieve sternal retraction.\n\nPostoperative care for study participants is standard of care. Additionally, as stated in the informed consent form, if participants become ill or injured as a direct result of participating in this study, necessary medical treatment will be available to them at no cost to them25.\n\nThe primary outcome measure will be the incidence of chronic post-sternotomy chest pain at 6 months following CABG with median sternotomy. However, we will also measure the incidence of CPSP at 3 and 12 months to determine the trajectory.\n\nSecondary outcomes (in addition to CPSP at 3 and 12 months) will include: Pain intensity (NRS scale) of sternal incision pain at rest and while coughing daily while in hospital until discharge and then at 1 week postoperatively (via telephone call). Analgesic consumption (morphine equivalents) daily while in hospital until discharge, and then at 1 week post-operatively and 3, 6, and 12 months post-operatively. Intra-operative data will include time from initiation to full retraction, latency from full retraction to sternal closure, width of sternal opening at full retraction. Pain quality (McGill Pain Questionnaire short form (MPQ-SF)), quality of life (SF-36 questionnaire), and pain interference with daily function (Brief Pain Inventory (BPI)) at 3, 6 and 12 months post-operatively. All assessments following discharge from the hospital will be via telephone calls from a research nurse blinded to randomization assignment. In-hospital assessments will also be done by a research nurse blinded to group assignment (Table 1). All data collection forms can be found as extended data25.\n\nPostoperative day 0 until patient discharge (d/c) from hospital\n\n*Completed if CPSP screen positive.\n\nBPI-SF-Brief pain inventory-short form; CPSP-Chronic post-sternotomy pain; D/C-discharge; DN4-Douleur Neuropathique 4; SF-MPQ-Short form McGill pain questionnaire; NRS-Numeric rating scale; POD0-Postoperative Day 0; SF36-36 item short form survey\n\nWe consider a 40% reduction in the incidence of CPSP at 6 months to be clinically significant. Since the prevalence of CPSP varies between 14–52% using current surgical methods and pain control strategies, and based on the work of Van Gulik et al. (2011)7 which observed a 35% incidence of CPSP at 12 months, we expect to see an incidence of 40% CPSP at 6 months, and expect to observe a 40% reduction (i.e., from 40% to 24%) with SLOW sternal retraction. Under these assumptions, we will require 132 patients per group to achieve 80% power at a two-sided alpha=0.05. We will conservatively increase our sample size to 158 per group to allow for up to 20% loss to follow-up (N=316) given the long duration of the follow-up period (i.e. 6 for the primary outcome and 12 months for secondary outcomes). A total of 10 additional patients will also be randomized to account for participants who may not receive the intervention (i.e., protocol non-compliance) or may be lost post-randomization due to surgical complications.\n\nPatients will be approached in the Pre-Anesthesia clinic by the research nurse prior to their surgery date. Eligible patients will be informed about the study and their eligibility to participate assessed. Informed consent will be obtained for all eligible and interested participants. If potential participants require more time to make a decision or would prefer to talk to others before making a decision, they can also take the forms home and if they do decide to participate, written informed consent will be obtained by research personnel on the day of surgery.\n\n\nAssignment of interventions: allocation\n\nA computer-generated randomization table will be prepared by the institutional biostatistician and patients randomly assigned in equal proportions to the standard or slow sternal retraction groups. Randomization will be unstratified in permuted blocks of randomly selected sizes of 4 or 8.\n\nThe institutional biostatistician will send the computer-generated randomization table directly to the departmental research secretary who has no involvement in the study other than preparation of the study envelopes. Sequentially numbered, sealed, opaque envelopes will be used to conceal the sequence allocation until the intervention is assigned in the operating room while the patient is under general anesthetic.\n\nA study nurse will enrol patients and assign them to the intervention based upon the consecutively numbered (and concealed) randomization assignment.\n\n\nAssignment of interventions: blinding\n\nThis is a double-blinded clinical trial (Participant, Outcomes Assessor). All OR staff, including surgeon(s) and anesthesiologist(s), will be unblinded during the surgery. Research staff who have no involvement in patient assessments will also be unblinded and in the OR to facilitate protocol compliance with the intervention and to measure the time to achieve sternal retraction.\n\n\nData collection and management\n\nDedicated, blinded research nurses will collect and compile the data. All assessment tools (described below) will be administered to the participant over the telephone at specified time-points (see Table 1). Kingston Health Sciences Centre biostatisticians will complete the majority of the data analysis. A research assistant will assist with the data cleaning and verification.\n\nThe Brief Pain Inventory-short form (BPI-SF) is a validated and reliable tool for measuring pain intensity and interference with daily activities. Scoring 1–10, where 1–4 = mild pain, 5–6 =moderate pain and 7–10= severe pain. Developed by the Pain Research Group of the WHO Collaborating Centre for Symptom Evaluation in Cancer Care26.\n\nThe 36-Item Short Form Health Survey (SF-36) is a validated and well-established tool which measures overall mental & physical health as a measure of Quality of life. Scores range from 0-100 where 0 is the lowest quality of life and 100 is the highest27.\n\nThe Short form-McGill pain questionnaire (SF-MPQ) will be used to evaluate the intensity & properties of the pain experience. The main component of the SF-MPQ consists of 15 descriptors (11 sensory; 4 affective) which are rated on an intensity scale as 0 = none, 1 = mild, 2 = moderate or 3 = severe. Three pain scores are derived from the sum of the intensity rank values of the words chosen for sensory, affective and total descriptors. The SF-MPQ also includes the Present Pain Intensity (PPI) index of the standard MPQ and a visual analogue scale (VAS)28.\n\nThe Douleur Neuropathique 4 (DN4) is a validated clinician-administered tool used to screen for patients experiencing neuropathic pain. The DN4 consists of 10 items split between 4 questions, where yes = 1, and no = 0. A score of 4 or greater indicates a patient likely experiencing neuropathic pain, with a sensitivity of 83% and a specificity of 90%29.\n\nThe Numeric Rating Scale (NRS) for pain is commonly used and validated tool used to measure pain intensity in adults. The NRS for pain uses an 11-point numeric scale (0–10) to represent a patient’s pain, where 0 represents one pain extreme (e.g., “no pain”) and 10 representing the other pain extreme (e.g., “worst pain imaginable”)30.\n\nAt the time of enrolment and randomization, participant contact information will be confirmed to ensure successful follow up. Permission to contact alternate contacts, family members, and/or the patients family physician will also be sought at the time of enrolment, to promote follow up if we are unable to contact the patient.\n\nData will be entered, verified, and stored in a password protected Excel spreadsheet. The study data will be identified only by a study ID number which will be linked to the patient’s identity only on a master (password-protected) spreadsheet stored separately from the data on a secure server. The de-identified data will be exported to SPSS for analysis purposes.\n\nA unique study ID number will be assigned to each participant. This study ID will be used on all data collection forms and will be linked with the true identity only on a master sheet which will be kept separate from the data in a locked cabinet. Data will be entered into electronic data spreadsheets and stored securely as described above. Only information directly surrounding the surgical procedure and/or relevant to this investigation will be collected. This will either come directly from the patient or from the medical record. Health information will only be used by the study investigators and will not be linked to any other sources. As stated above the information will be identified only by a study ID number. This data may be used for presentation or publication, but no identifiable information will be used. Only principal investigators or their delegates at KHSC will have access to this information. Delegates will include the research nurses who will collect and compile the data, the KHSC biostatisticians who will complete the majority of the data analysis, a research assistant who will assist with the data cleaning and verification. Data will be stored securely for 25 years as required, following which it will permanently destroyed.\n\n\nStatistical methods\n\nData will be imported into IBM SPSS (version 26 for Windows, Armonk, New York, 2018) for statistical analysis. Data will initially be described using frequencies and percentages, means and standard deviations or medians and quartiles as appropriate, to examine the data and identify any inappropriate values. The normality of the continuous data will be assessed using the Shapiro-Wilk test. The two groups will them be compared using the Pearson chi-square test (or Fisher’s Exact test in the event of small cells) for the primary outcome of the incidence of CPSP at 6 months, as well as at 3 and 12 months. Secondary outcomes will be similarly assessed using Chi-square tests for categorical outcomes, and independent samples t-tests (or the Mann-Whitney U) for continuous outcomes. Multivariable logistic regression analyses will be conducted to determine which factors are associated with the development of CPSP. Variables entered into the model will include those with a p-value of <0.15 in bivariate analyses, as well as the assigned group. Statistical comparisons of the secondary outcomes will be considered exploratory and will not be used to assert the efficacy of the treatment in the absence of a statistically significant benefit in the primary outcome. A p-value of <0.05 will be considered as criteria for statistical significance, and no adjustment will be made for multiple comparisons.\n\nIntent to treat analyses will not be conducted. Only data for those participants who completed the trial will be included in the analysis, with no corrections made for missing values.\n\n\nOversight and monitoring\n\nComposition of the coordinating centre and trial steering committee\n\nDr. Tarit Saha is the Principal Investigator (MD, FRCPC) and cardiac anesthesiologist at Queen’s University & affiliated teaching hospitals. Dr. Saha is a new investigator despite the fact that he has been an attending cardiac anesthesiologist at Kingston area hospitals since 2004.\n\nDr. Dimitri Petsikas is the Co-Principal Investigator (MD, FRCSC) and a cardiac surgeon at Queen’s University and affiliated teaching hospitals.\n\nComposition of the data monitoring committee, its role and reporting structure\n\nOur Data Safety Monitoring Committee is composed of Dr. Ken Reid, Thoracic Surgeon, and Dr. Catherine McLellan, Cardiologist. None of them have any direct contact with the study. This committee will review adverse outcomes in a blinded fashion half of the way through enrolment.\n\nAdverse event reporting and harms\n\nAll serious adverse events (SAEs) will be reported to Principal Investigator immediately and the Research Ethics Board (REB) will be notified. Adverse events also be reported as per institutional policy and will be followed to resolution or stabilization.\n\nA trial audit by our DSMB members will be conducted mid-trial to investigate relevant SAEs in both groups to determine if it is safe to continue.\n\nPlans for communicating important protocol amendments to relevant parties (e.g. trial participants, ethical committees)\n\nAny protocol modifications or deviations will be reported to all investigators and will be approved by the REB. The trial Registry will be updated accordingly.\n\n\nDissemination plans\n\nThe results of our trial will be disseminated through national and/or international conferences, publications, as well as through discussions with cardiac surgeons at other centers.\n\n\nTrial status\n\nThe current investigation is near completion, having randomized and collected primary endpoint data on all patients enrolled. Data collection of remaining secondary endpoints will be completed in early 2021. This is protocol version 1.2.\n\n\nData availability\n\nNo data are associated with this article.\n\nQueen’s University Dataverse: Does the Speed of Sternal Retraction during Coronary Artery Bypass Graft Surgery affect Postoperative Pain Outcomes? A Randomized Controlled Trial Protocol https://doi.org/10.5683/SP2/LQC1JH25\n\nThis project contains the following extended data:\n\n- Consent form\n\n- Post operative data collection form\n\n- Follow up questionnaire (SF-36)\n\n- Follow up questionnaire (BPI, DN4, MPQ-SF)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nThe authors would like to thank Andrew Day for generating the randomization sequence, and Wilma Hopman for her contributions to the statistical methods.\n\n\nReferences\n\nLiu SS, Wu CL: Effect of postoperative analgesia on major postoperative complications: a systematic update of the evidence. Anesth Analg. 2007; 104(3): 689–702. PubMed Abstract \| Publisher Full Text\n\nLi Z, Armstrong EJ, Parker JP, et al.: Hospital variation in readmission after coronary artery bypass surgery in California. Circ Cardiovasc Qual Outcomes. 2012; 5(5): 729–37. PubMed Abstract \| Publisher Full Text\n\nJensen TS, Turner JA, Wiesenfeld-Hallin Z, et al.: Proceedings of the 8th World Congress on Pain. Progress in Pain Res Manag Seattle IASP Press, 1997; 8: 231–240. Reference Source\n\nRowe MA, King KB: Long-term chest wall discomfort in women after coronary artery bypass grafting. Heart Lung. 1998; 27(3): 184–8. PubMed Abstract \| Publisher Full Text\n\nSteegers MAH, van de Luijtgaarden A, Noyez L, et al.: The role of angina pectoris in chronic pain after coronary artery bypass graft surgery. J Pain. 2007; 8(8): 667–73. PubMed Abstract \| Publisher Full Text\n\nCantero C, Carolina G, Matute P, et al.: Eur J Anaesthes conference proceedings EUROANAESTHESIA. 2011; 28: 193–194.\n\nvan Gulik L, Janssen LI, Ahlers SJGM, et al.: Risk factors for chronic thoracic pain after cardiac surgery via sternotomy. Eur J Cardiothorac Surg. 2011; 40(6): 1309–13. PubMed Abstract \| Publisher Full Text\n\nCenters for disease control: US National Hospital discharge survey. 2009. Reference Source\n\nCanadian Institutes for Health Information: Health indicators interactive tool. (Information from the discharge abstracts database Jan 1-Dec 31, 2009). Accessed Nov 25, 2012. Reference Source\n\nKissin I, Gelman S: Chronic postsurgical pain: still a neglected topic? J Pain Res. 2012; 5: 473–89. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKatz J, Seltzer Z: Transition from acute to chronic postsurgical pain: risk factors and protective factors. Expert Rev Neurother. 2009; 9(5): 723–44. PubMed Abstract \| Publisher Full Text\n\nMailis A, Chan J, Basinski A, et al.: Chest wall pain after aortocoronary bypass surgery using internal mammary artery graft: a new pain syndrome? Heart Lung. 1989; 18(6): 553–8. PubMed Abstract\n\nKing KM, Parry M, Southern D, et al.: Women’s Recovery from Sternotomy-Extension (WREST-E) study: examining long-term pain and discomfort following sternotomy and their predictors. Heart. 2008; 94(4): 493–7. PubMed Abstract \| Publisher Full Text\n\nKalso E, Mennander S, Tasmuth T, et al.: Chronic post-sternotomy pain. Acta Anaesthesiol Scand. 2001; 45(8): 935–9. PubMed Abstract \| Publisher Full Text\n\nLamacraft G: The link between acute postoperative pain and chronic pain syndromes. Southern African Journal of Anaesthesia and Analgesia. 2012; 18(1): 45–50. Publisher Full Text\n\nBolotin G, Buckner GD, Campbell NB, et al.: Tissue-Disruptive Forces during Median Sternotomy. Heart Surg Forum. 2007; 10(6): E487–92. PubMed Abstract \| Publisher Full Text\n\nBolotin G, Buckner GD, Jardine NJ, et al.: A novel instrumented retractor to monitor tissue-disruptive forces during lateral thoracotomy. J Thorac Cardiovasc Surg. 2007; 133(4): 949–54. PubMed Abstract \| Publisher Full Text\n\nMoustafa MA, Abdelsamad AA, Zakaria G, et al.: Minimal vs median sternotomy for aortic valve replacement. Asian Cardiovasc Thorac Ann. 2007; 15(6): 472–5. PubMed Abstract \| Publisher Full Text\n\nCandaele S, Herijgers P, Demeyere R, et al.: Chest pain after partial upper versus complete sternotomy for aortic valve surgery. Acta Cardiol. 2003; 58(1): 17–21. PubMed Abstract \| Publisher Full Text\n\nAris A, Cámara ML, Montiel J, et al.: Ministernotomy versus median sternotomy for aortic valve replacement: a prospective, randomized study. Ann Thorac Surg. 1999; 67(6): 1583–7. PubMed Abstract \| Publisher Full Text\n\nBauer M, Pasic M, Ewert R, et al.: Ministernotomy versus complete sternotomy for coronary bypass operations: no difference in postoperative pulmonary function. J Thorac Cardiovasc Surg. 2001; 121(4): 702–7. PubMed Abstract \| Publisher Full Text\n\nMazzeffi M, Khelemsky Y: Poststernotomy pain: a clinical review. J Cardiothorac Vasc Anesth. 2011; 25(6): 1163–78. PubMed Abstract \| Publisher Full Text\n\nCasey KL: Concepts of pain mechanisms: the contribution of functional imaging of the human brain. Prog Brain Res. 2000; 129: 277–87. PubMed Abstract \| Publisher Full Text\n\nJulius D, Basbaum AI: Molecular mechanisms of nociception. Nature. 2001; 413(6852): 203–10. PubMed Abstract \| Publisher Full Text\n\nPetsikas D, Stewart C, Phelan R, et al.: Does the Speed of Sternal Retraction during Coronary Artery Bypass Graft Surgery affect Postoperative Pain Outcomes? A Randomized Controlled Trial Protocol. Scholars Portal Dataverse, V1. 2021. http://www.doi.org/10.5683/SP2/LQC1JH\n\nCleeland CS, Ryan KM: Pain assessment: global use of the Brief Pain Inventory. Ann Acad Med Singap. 1994; 23(2): 129–38. PubMed Abstract\n\nWare JE Jr, Sherbourne CD: The MOS 36-item short-form health survey (SF-36). I. Conceptual framework and item selection. Med Care. 1992; 30(6): 473–83. PubMed Abstract \| Publisher Full Text\n\nMelzack R: The short-form McGill Pain Questionnaire. Pain. 1987; 30(2): 191–7. PubMed Abstract \| Publisher Full Text\n\nBouhassira D, Attal N, Alchaar H, et al.: Comparison of pain syndromes associated with nervous or somatic lesions and development of a new neuropathic pain diagnostic questionnaire (DN4). Pain. 2005; 114(1–2): 29–36. PubMed Abstract \| Publisher Full Text\n\nDownie WW, Leatham PA, Rhind VM, et al.: Studies with pain rating scales. Ann Rheum Dis. 1978; 37(4): 378–81. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "86550", "date": "11 Jun 2021", "name": "Amr Arafat", "expertise": [ "Reviewer Expertise Cardiothoracic Surgery" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors submitted a proposal for a randomized clinical trial comparing the effect of the speed of sternal retraction on postoperative pain after coronary artery bypass grafting. The topic is interesting and was not thoroughly evaluated before. Here are my comments and questions related to this proposal:\nTitle:\nThe title is suitable and clearly defines the research topic.\nAbstract:\nPlease state the objective of your research at the end of the background.\n\nIn the method section, please explain what do you mean by a double-blind study in the abstract?\n\nThe introduction and objectives clearly stated the research gap and question.\nDesign:\nSuitable to answer the research question.\n\nInclusion and exclusion criteria were clearly defined, but please give examples of conditions leading to the inability to perform postoperative assessments.\n\nThe authors did not clarify if the slow method will be used every time the sternal retractor needs to be reinserted. Will you exclude patients who required sternal opening more than the pre-defined diameter?\n\nSample size calculation is adequate.\nStatistical analysis:\n\nLongitudinal analysis for repeated measures is required.\n\nMethod sections were clearly described and presented.\nReferences are old. Please cite recent references or state whether there are no recent publications on this topic.\nLastly, I’d like to congratulate the authors on this well-written proposal.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [] }, { "id": "88529", "date": "13 Jul 2021", "name": "Ferhat Borulu", "expertise": [ "Reviewer Expertise Cardiovascular surgery", "vascular surgery" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nFirst of all, I would like to congratulate the authors for this study, which considers it to be beneficial for postoperative sternotomy pain, which is neglected by most cardiac surgeons.\nI'm quite curious about the authors' approach to some issues:\nIs it considered to establish a relationship between pain and the drains used? Because especially thoracic drains have a highly increasing aspect for postoperative pain. Is there a plan for the effect of drains on the need for pain relief?\n\nInformation should be given about the retractors to be used in the preparation of the internal thoracic artery. The types of retractors used for this procedure may also contribute to sternum pain.\n\nAll the details of the study are presented very well. However, it will be very difficult to involve so many patients in working with phones for quite a long time. How will the motivation of the patient and the work team be provided in this regard? It seems to be quite difficult to follow up, especially with the phone.\nSince I do not have enough knowledge about statistics, there is no statistical part in my analysis.\nI would like to wish success to the working team in advance.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-248
https://f1000research.com/articles/8-958/v1	24 Jun 19	{ "type": "Study Protocol", "title": "aDolescents gEnder surVey, rEsponsible coupLes evaluatiOn, and capacity building Project in India (DEVELOP): a study protocol", "authors": [ "Anand Ahankari", "Mark Hayter", "Clare Whitfield", "Parveen Ali", "Sneha Giridhari", "Shruti Tambe", "Pratyush Kabra", "Kranti Rayamane", "Pavel Ovseiko", "Clare Whitfield", "Parveen Ali", "Sneha Giridhari", "Shruti Tambe", "Pratyush Kabra", "Kranti Rayamane", "Pavel Ovseiko" ], "abstract": "Gender-based violence against women is a serious concern in India. This affects the health and wellbeing of victims and their dependents. Published evidence has documented a variety of reasons for such violence in Indian societies, paving a pathway to design, implement, and evaluate intervention models to address this issue. DEVELOP is a research study designed by UK and Indian research teams to plan future projects to address gender-based discrimination and violence against women and girls in India. This study protocol provides detailed information on the objectives, research methods, data collection, storage, analysis, and dissemination plans of the DEVELOP. The first component is a survey of adolescent boys and girls from rural areas of Maharashtra state of India to understand their gender equality related knowledge and beliefs. The insight gathered will be used to design interventions targeted at adolescent populations through future research and development programmes. Secondly, an evaluation of the ‘Responsible Couples’ project will be conducted to assess its success and challenges, and to design suitable programme activities and models. The ‘Responsible Couples’ project is implemented in 40 villages of Maharashtra state to improve relationships in married couples, prevent violence against women, intervene during violence, and to provide support services for women and their family members. Research findings will be disseminated though public engagement events in India, international conferences, and peer reviewed publications. This will impact on the policy and work models of Indian partners to plan future project investments. Research findings will be also useful for local government authorities and non-government agencies striving to advance gender equality.", "keywords": [ "Gender", "Maharashtra", "India", "Adolescent", "Violence", "Evaluation" ], "content": "Introduction\n\nGender-based violence against women is a fundamental violation of women’s human rights, health, and wellbeing1. Globally, intimate partner violence (IPV) is the most common form of gender-based violence against women1. A recent study from south India reported that over 50% of women had experienced physical domestic violence ever, and 27% faced physical domestic violence in the past six months2. The majority of female victims are married to the perpetrator and underreporting of IPV is a known phenomenon3,4. IPV is linked to a range of factors including alcohol addiction, financial debt, cultural and social acceptance of violence, and childhood trauma/exposure to violence4. These factors are also linked to gender-based discrimination, adversely affecting girls’ and women’s health and well-being, their ability to continue their education, choose a career, make informed reproductive decisions, and achieve financial independence. Adverse effects of gender-based discrimination on girls’ and women’s health and wellbeing are particularly high in deprived communities3,4.\n\nAlthough the need to reduce gender-based violence and empower women in India is widely recognised, there is very limited information available on gender equality-related knowledge, attitudes, and behaviours among Indian adolescents, who are an important age group to target with public health and development interventions. Moreover, traditionally, interventions to reduce gender-based violence and empower women were focused on women, and men were predominantly left out of such interventions. Overall, there is very limited research on the effectiveness of gender-based violence reduction interventions.\n\nWith financial and technical support from the SWISSAID5, Halo Medical Foundation (HMF), an NGO working in Maharashtra state of India, has developed and is currently implementing the ‘Responsible Couples’ intervention to address gender-based violence against women by educating men, supporting women, and providing village infrastructural facilities to create healthy relationships and violence-free communities6. The ‘DEVELOP’ project seeks to increase understanding of gender equality-related knowledge, attitudes, and behaviours among Indian adolescents, as well as evaluate the ‘Responsible Couples’ intervention. The project is planned to be conducted in Maharashtra state of India in 2019. This paper is a study protocol of the DEVELOP providing detailed information on study objectives, research methods of two main components of the project, data storage, handling, and dissemination plans.\n\n\nProtocol\n\nTo conduct feasibility and capacity building work in India to support future research and development projects in gender equality.\n\nTo inform the development of a survey tool measuring gender equality related knowledge, attitudes, and behaviours in Indian adolescents.\n\nTo conduct a qualitative evaluation of the current gender-based violence reduction intervention–the ‘Responsible Couples’ project in the rural region of Maharashtra state of India.\n\nThis study has two components as outlined below to achieve the research objectives. The project team structure and partners are outlined in Figure 1.\n\nHMF, Halo Medical Foundation; FGD, focus group discussion.\n\nI. Quantitative component - a questionnaire survey of the gender equality beliefs and attitudes of minimum 1000 young people (male and female) aged 16 to 19 from 70 villages of Maharashtra state, India.\n\nII. Qualitative component - focus group discussions with the local gender equality promotion groups in 12 villages of Maharashtra state to explore their views on the implementation of the ‘Responsible Couples’ project, what challenges they are experiencing in their gender equality work, what they feel about the impact of their work, and how the project can be improved further.\n\nI. Quantitative component\n\nThe project field consists of 70 villages from HMF’s area of work, located in Osmanabad district of Maharashtra state of India. The study will invite all adolescents aged 16 to 19 from 70 villages to participate in the project by completing a questionnaire. These villages define HMF’s current geographic scope based on funding allocated by the SWISSAID to work in Maharashtra. Future programme and intervention development work will involve the same villages; therefore, these were considered in the DEVELOP research project.\n\nEach village has a member of HMF staff linked to it. This individual will help distribute written information about the project (in the form of a leaflet, Supplementary File 1, Extended data7) in each village at least two weeks before any data is collected. Leaflets will be distributed to cover all community areas in each village. There are also field staff such as healthcare workers in project area. These workers are also able to provide any verbal description of the project if required at this point in time. They will also orally inform of the date of the data collection event. All staff members including field staff received necessary research and ethics training in February and April 2019 and are supervised by a senior research co-ordinator on a daily basis with additional support from a project manager based at HMF.\n\nData collectors will then visit the village for one day of data collection. They will set up their station at a village health centre, school or other locally available building/resource, where interested adolescents will be invited to visit to find out more about the project. Written and verbal explanations will be given to those who have not seen the previous information sheet (Supplementary 1, Extended data)7.\n\nThe study will be open to boys and girls aged 16 to 19 only. Participants should be able to read and write in local language (Marathi) in order to understand the project information sheet and complete the data collection form independently. Data collection documents were translated from English to Marathi by a project manager in the first instance, and the translation was verified by authors with bilingual proficiency (AA and SG). The final data files were reviewed several times to ensure its accuracy. The adolescents who agree to participate will be given a questionnaire, pen, and sealable envelope. The on-site data collection staff will address any queries, if asked by participants.\n\nThe adolescent survey will be conducted to collect a minimum of 1000 questionnaires. This number is based on discussions with project partners to ensure that results from this study will be applicable to rural areas of Maharashtra state. This is also the maximum number of participants this project could achieve considering available resources. A similar strategy was used to conduct research in this field area involving adolescent girls8.\n\nIn order to collect representative data by age and gender, a stratified sampling technique will be used. In total, a minimum 1000 questionnaires will be completed, including 125 from each age group16,17,18,19 and 500 from each gender. This is the ideal sample scenario; however, no participants will be turned away on a data collection day even if the said number has already been achieved.\n\nA survey questionnaire in the local language (Marathi) will be used to collect data from adolescents. An English version of the questionnaire is available as Extended data7. The tool is developed based on a validated and published questionnaire9,10, which was used to study gender equality among Indian adolescents9. The questionnaire was iteratively revised and improved within the team, discussed with partners, and then piloted and validated prior to administration. The outlined process was completed through focus group discussions and a testing phase involving adolescent boys and girls at the HMF training centre in March 2019. The feedback from the discussions was included, and minor changes were made mainly on the structure of the data collection form. No major changes such as question re-structuring were required. The questionnaire used in this study has a section on basic demographics (12 questions) and then three individual sections to measure knowledge (nine statements), attitudes (six statements) and behaviours (seven statements) related to gender equality (Supplementary 2, Extended data)7. The gender equality score for each participant will be calculated for the three sections of the questionnaire (knowledge, attitudes and behaviour) using the following method. For each statement, the score will range from zero to two. Those who agreed with a given statement, indicating a lack of support for gender inequality, receive a score of zero. Those who partially agree receive a score of one, and those who disagreed receive a score of two, indicating support for gender equality. The total score will be calculated for each completed questionnaire by adding the score for all 22 statements. Total scores for each questionnaire will range from a low of zero (highly gender inequitable) to a high of 44 (highly gender equitable).\n\nTo ensure confidentiality, questionnaires will be completed in an area of the village hall/health centre that affords privacy. The completed questionnaires (in sealed envelopes) are then placed in a box by participants as they leave the hall/data collection centre. The overall data collection will be supervised by a qualified member of HMF staff.\n\nThe questionnaire will not collect any personal identifiable information such as name, home address, or contact details. Both the study information sheet and the questionnaire will include information assuring the participants of confidentiality and how the data will be used. Participants will not be identified or identifiable through reports or publications and only the research team will have access to the data. All data in India will be stored on a password-protected computers and encrypted USB devices and will only be accessible to the project and research teams. The survey data will be moved to the University of Hull, UK in a Microsoft Excel file, stored on a secure server and used for analysis purposes. A member of the research team will access the data stored at HMF office in person and will upload the data remotely to the University of Hull online storage server using secured login details. This will be verified by another team member to ensure that all data are safely moved to the University of Hull online storage system. The data will be stored for five years following the project completion.\n\nIncomplete questionnaires will be discarded from analysis and stored for auditing purposes. Survey responses will be analysed in Stata (StataCorp, College Station, Texas, USA) and/or SPSS (IBM) using descriptive statistics, tests of statistical significance, and reliability coefficients. If data permits, then additional analysis to compare results across villages/blocks/districts would be also conducted. Results will be reported in line with STROBE guidelines11.\n\nII. Qualitative component\n\nThe ‘Responsible Couples’ project is currently being implemented in 40 villages of Osmanabad district of Maharashtra state, India. Each village has one local group comprised of 15 to 20 village members, who are working towards gender equality in their community. Group members have been trained by subject experts and receive mentoring support from HMF project implementation staff. The groups provide support and facilitate access to victims of IPV and intervene to prevent violence against women, focussing on those who are married and living with their husband and/or in-laws. Importantly, as part of HMF’s work to support research development, these groups have been involved in the inception of the current research study.\n\nFrom a list of 40 villages, 12 villages will be randomly selected for focus group discussions. In order to ensure random selection, all villages will be randomly numbered by a project manager based in India, and a total of 12 numbers will be selected by a member of the research team (AA, based in the UK). The process will be completed over email to record the process. At least two weeks before focus group discussions are held, members of the research/project team will convene meetings with the members of the village gender equality groups in the selected villages to describe the project and answer questions. To accompany the verbal description of the project, each group member will receive an information sheet (Supplementary 3, Extended data)7. Only existing members of the village gender equality groups in the selected villages will be invited to participate in focus group discussions.\n\nTwelve focus group discussions are expected to provide sufficient insights into the implementation of the ‘Responsible Couples’ intervention across 40 participating villages. It is expected that up to 10 members from each village will participate in each focus group discussion. Based on our series of consultations with partners, field visits, interactions with beneficiaries, the proposed 12 focus group discussions is expected to be sufficient to achieve data saturation.\n\nA discussion guide in a local language (Marathi) will be used to facilitate focus group discussions. An English version is included as Extended data (Supplementary 4)7. Data collection documents were translated from English to Marathi by a project manager at first instance and were verified by authors with bilingual proficiency (AA and SG). The final data files were reviewed several times to ensure its accuracy. The discussion guide was revised iteratively within the team, discussed with partners, and then piloted prior to research use. The interview guide was used to conduct discussions in two villages from the project areas where its structure, questions were tested. This was attended by a project manager and a senior research co-ordinator to provide feedback to investigators based in India and the UK. No amendments to the guide were required.\n\nFocus group discussions will be conducted, transcribed, and translated by two experienced facilitators with bilingual skills (Marathi and English) under the supervision of the research team. The qualitative data will include information on the views of village level groups on: the implementation of the ‘Responsible Couples’ project; what challenges they are experiencing in their gender equality work; what they feel about the impact of their work; and how the project can be improved further.\n\nNo personal information will be collected during the focus groups. The information sheet will include assurances on confidentiality and that no identifiable data will be used in reports and publications. Only the research team will have access to the data. All data in India will be stored on password-protected computers and encrypted USB devices and will only be accessible to the research team. Once research activities are completed in India, the focus group discussion data will be moved to the University of Hull, UK in Microsoft Word and PDF files with the audio recorded discussion files, stored on a secure server and used for analysis purposes, as described for the quantitative data.\n\nQualitative data from the focus group discussions will be analysed thematically and organised in NVivo (QSR International). Two researchers will independently code data and synthesise the findings into themes. They will then meet to discuss areas of agreement and disagreement and reach consensus on the coding tree, illustrative quotations, and interpretation. Results will be reported in line with COREQ guidelines12.\n\nThe study has been approved by the Faculty of Health Sciences Ethics Committee, University of Hull, UK (approval reference number- FHS125 issued on 3rd April 2019) and the ethics committee of the Ashwini Rural Medical College, Hospital and Research Centre, Solapur, Maharashtra, India (approval reference number- ARMCH/IECHR/03/2019 issued on 15th March 2019). All survey participants will give individual oral informed consent before completing the questionnaire. The oral informed consent was preferred in this survey to ensure full confidentiality of participating adolescents. Written consent requires basic details such as name, address with further requirements of anonymisation. These details are not requested on the questionnaire and therefore oral informed consent is deemed sufficient from a willing participant before handling over the questionnaire. As a result of this, no personal information on participating adolescents such as name or address are collected at any point of time. All focus group participants will give a collective written informed consent before participating in focus group discussions. This strategy was decided following consultation with our project partners. At the start of the session, a collective signed consent form will be obtained. In India, the provision of a personal signature on a form is regarded with some suspicion and the collective form alleviates this. Further, all eligible participants are active members of the village level group and regularly meet for monthly meeting and thus are aware about collective signatures as a part of on-going project activities. This also allows the data collection team to collect the signatures of willing participants where individual names, addresses and contact details were not collected. The consent form is provided as Extended data (Supplementary 5)7.\n\nThe results of the study will be disseminated via local, regional, and national dissemination events, online video and blogs, peer-reviewed publications, and presentations at international conferences.\n\nThe DEVELOP project duration is from December 2018 to July 2019. The study is currently in the data collection phase (planned to take place from April to July 2019).\n\n\nConclusions\n\nTo the best of our knowledge, this will be the first survey from the Maharashtra state of India, and one of the largest surveys, measuring gender equality-related knowledge, attitudes, and behaviours among Indian adolescents. The survey findings will generate new valuable insights into how adolescent groups could be engaged in the future to improve gender equality in Indian communities.\n\nThe qualitative evaluation will inform the implementation of the ‘Responsible Couples’ intervention and strategies to improve it. It will also have policy implications for HMF, SWISSAID, and other organisations seeking to reduce gender-based violence and empower girls and women in Maharashtra and other states of India. Considering diverse Indian culture, practices, and beliefs, the study results may not be completely generalizable beyond the population studied.\n\nThe DEVELOP project will contribute to research capacity building and evidence-based practice in a limited resource setting. The project will provide opportunities to train and engage over 10 researchers and practitioners in Maharashtra state of India to improve their knowledge, develop new research skills, and enhance their experience of collaborating with international partners. It is expected that the project will help partners involved from India and the UK to develop further research on the adoption, implementation, and scale-up of evidence-based gender equality interventions in Maharashtra and other Indian states and territories.\n\n\nData availability\n\nNo underlying data are associated with this article.\n\nFigshare: ADolescents GEnder SurVey, REsponsible CoupLes EvaluatiOn, and Capacity Building Project in India (DEVELOP): A study protocol. https://dx.doi.org/10.6084/m9.figshare.8256050.v17\n\nThis project contains the following extended data:\n\n- Supplementary Files 1 to 5.pdf (participant information sheet for questionnaire, questionnaire in English, participant information sheet for focus group discussion, focus group discussion guide in English, focus group discussion consent form)\n\n- DEVELOP_Survey questionnaire Marathi.pdf (questionnaire in Marathi)\n\n- DEVLOP_FGD Guide Marathi.pdf (focus group discussion guide in Marathi)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Grant information\n\nThe DEVELOP project is formulated following a travel grant awarded to Prof Mark Hayter and Dr Anand Ahankari in May 2018 from the Global Challenges Research Fund (GCRF) allocated to the University of Hull, UK. The project received a dedicated grant in November 2018 from the Global Challenges Research Fund (GCRF) allocated to the University of Hull, UK. Dr Pavel Ovseiko is supported by the European Union’s Horizon 2020 research and innovation programme award STARBIOS2 [709517] and by the National Institute for Health Research, grant NIHR Oxford Biomedical Research Centre [BRC-1215-20008] to the Oxford University Hospitals NHS Foundation Trust and the University of Oxford.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nFirstly, we thank the Research Facilitation Office of the University of Hull, UK for providing financial support through the Global Challenge Research Fund Pump Priming Grant initiative (GCRF Pump Priming Award 2018-19). We acknowledge the support received from the SWISSAID India liaison office, and Halo Medical Foundation, India for offering access to project area and sharing their experiences to conceptualise this study and also the grant application. We thank Dr Shashikant Ahankari (President, HMF) and Ms Kavita Gandhi (Country Representative, SWISSAID India) for their support and guidance. We also thank Mr Milind Hardikar (SWISSAID India) for his support in finance monitoring and project administration.\n\n\nReferences\n\nWorld Health Organisation: Violence against women. [accessed on 5 May 2019]. Reference Source\n\nRocca CH, Rathod S, Falle T, et al.: Challenging assumptions about women's empowerment: social and economic resources and domestic violence among young married women in urban South India. Int J Epidemiol. 2009; 38(2): 577–585. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFulu E, Warner X, Miedema S, et al.: Why Do Some Men Use Violence Against Women and How Can We Prevent It? Quantitative Findings from the United Nations Multi-country Study on Men and Violence in Asia and the Pacific. Bangkok: UNDP, UNFPA, UN Women and UNV, 2013. Reference Source\n\nInternational centre for research on women: Men, Masculinity andDomestic Violence in India. [accessed on 10 May 2019]. Reference Source\n\nSWISSAID, India. [accessed on 6 June 2019]. Reference Source\n\nHalo Medical Foundation, India. [accessed on 6 June 2019]. Reference Source\n\nAhankari A, Hayter M, Whitfield C, et al.: ADolescents GEnder SurVey, REsponsible CoupLes EvaluatiOn, and Capacity Building Project in India (DEVELOP): A study protocol. 2019. http://www.doi.org/10.6084/m9.figshare.8256050.v1\n\nAhankari AS, Myles PR, Fogarty AW, et al.: Prevalence of iron-deficiency anaemia and risk factors in 1010 adolescent girls from rural Maharashtra, India: a cross-sectional survey. Public Health. 2017; 142: 159–166. PubMed Abstract \| Publisher Full Text\n\nAchyut P, Bhatla N, Khandekar S, et al.: Building Support for Gender Equality among Young Adolescents in School: Findings from Mumbai, India. ICRW, New Delhi. [accessed on 16 May 2019]. Reference Source\n\nNanda G: Compendium of Gender Scales. Washington, DC: FHI 360/C-Change 2011. [accessed on 16 May 2019]. Reference Source\n\nvon Elm E, Altman DG, Egger M, et al.: The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement: guidelines for reporting observational studies. J Clin Epidemiol. 2008; 61(4): 344–9. PubMed Abstract \| Publisher Full Text\n\nTong A, Sainsbury P, Craig J: Consolidated criteria for reporting qualitative research (COREQ): a 32-item checklist for interviews and focus groups. Int J Qual Health Care. 2007; 19(6): 349–357. PubMed Abstract \| Publisher Full Text" }	[ { "id": "53221", "date": "16 Sep 2019", "name": "Prashanth N Srinivas", "expertise": [ "Reviewer Expertise Public health", "realist evaluation", "health policy & systems research methods" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper is a study protocol of research proposed in Osmanabad district of Maharashtra, India to improve evidence-base for understanding and acting upon gender-based violence (GBV). The study seeks to understand gender equality related knowledge, attitudes and practices among adolescents on one hand, and evaluate an ongoing intervention to reduce gender-based violence, called “Responsible Couples” (RC). The authors make a strong case for the need for such evidence (from adolescents, where interventions targeting the problem ought to begin) as well as the relative lack of effectiveness evidence from interventions to reduce GBV. Authors indicate that this could be the first of its kind large survey on this topic in Maharashtra, and perhaps among Indian adolescents. In addition, their remarks on the research capacity-building and academic exchange involved are also noted with appreciation.\n\nBackground The background provides a published estimate of domestic violence (DV) ever and DV in the past 6 months. The study cited surveyed households in one city of south India, whereas the background states this as being an estimate for south India, which may not be an appropriate representation of the original study’s findings.\n\nImproving coherence through more contextual information and clarifying objectives A project goal is separately provided in addition to the research objectives. The two research objectives focus on (1) survey tool development for adolescents, and (2) evaluation of the RC program. The project goal is quite broad and both objectives fit within this goal, but it is unclear how these two objectives come together. Is there any specific reason these two objectives have been chosen? What was the rationale for choosing this particular intervention? Perhaps this is related to specific contextual information that may not be part of the protocol. Researchers could consider a section that describes the particular setting where the study is being planned as well as perhaps a section that describes work leading up to the study to provide the reader an understanding of the logic/reasoning in choosing these two objectives in order to work towards the project goal.\n\nWhile the background indicates that the authors will undertake a survey among adolescents (and indeed the data collection and subsequent activities confirm this, the first of the two objectives that include this appears to stop short at “…inform(ing) the development of a survey tool”. This gives an impression of this being a tool-development/feasibility study only. Clarify.\n\nSelection and recruitment\nThe project identifies 70 villages which correspond to the area of work of partner NGO as being the study area. It is unclear from the information provided how much of the district this covers, and what the original logic is for the partner NGO to choose these villages. Since a survey is being planned, the reporting of its findings will depend on the overall choice of study site and sampling and hence further information has to be provided to enable an understanding of the study area. Some information on the larger context in terms of how this district compares with other districts in the state with respect to GBV are useful (if available). An estimate of what proportion of the district is covered by these villages is also useful.\n\nAuthors indicate that the age of eligible respondents for the survey shall be 16 to 19. How will this be verified (if at all)? Will this be based on self-reporting of age? Or will researchers have an estimate/list from prior work?\n\nWill estimates/identity of adolescents in these villages be available to researchers from the existing work of partner NGO? If yes, specify how this data will be dealt with and used/not used. Authors may consider comparing their village-wise sample with adolescents in the village to come up with an estimate of their overall survey coverage among adolescents.\n\nIf adolescents who are unable to read/write turn up, what will the team do? There are both technical and ethical implications of this decision to exclude this group, even if the numbers in this group are small. More so given that educational status has been declared in the background to influence GBV.\n\nSample size\nAuthors state that one of the reasons for choosing a sample size of 1000 is “…to ensure that results from this study will be applicable to rural areas of Maharashtra state.”. This will need to be explained clearly. Specifically, the concern is with respect to (a) how would any sample size defined within a set of villages in one district of the state “…be applicable” to the entire state? Perhaps the authors seek to achieve analytical generalisability of their findings through mixed-method/qualitative inquiry and demonstrate the relative similarity of few/many/all other districts to this district with respect to the area of inquiry, but this cannot be based on a particular sample size in my assessment. It is also noted that another logic to arrive at the size has been the resource availability. On the contrary, could an effort at achieving a sampling strategy of smaller numbers of household visits be used to further minimize sample numbers if needed? I understand that the work may have already begun, in which case, authors need to clarify this section appropriately.\n\nAuthors mention a stratified sampling approach, but the survey is being conducted by invitation to a “station” at each village. How will the 125 per each age-stratum and 500 from each gender be achieved if there is a patterning in age of children who turn up at the station (for instance, older boys being less “shy” of such a station, or for instance if some stations for reasons unknown are not acceptable/accessible for particular age/gender respondents). Given that the researchers do not identify specific strategies to ensure the sample across strata, this stratified sampling appears more to be a desire at ensuring distribution of sample across all age-groups rather than an actual sampling strategy? Clarify.\n\nRC program evaluation\nIn terms of improving the coherence between these 2 objectives, and also given that both are being implemented in the same district, it is useful to understand if the 40 villages where RC will be evaluated are a sub-set of the 70 where the quantitative survey will be conducted?\n\nThe RC group in each village: Are these volunteers or paid staff of the project? Clarify.\n\nGiven that the objective is to evaluate an intervention which has multiple components, an overall evaluation approach to the intervention is missing. Partly, this could be dealt with by providing more details of how the qualitative data will be analyzed from the FGDs. Also to some extent, the evaluation will only engage with relative insiders (the village level RC group) and will not include perspectives of those who \"received\" the intervention, a limitation. Authors could consider including a clear schematic/narrative on how the intervention is expected to work outlining the various intervention inputs, assumptions being made and how it is linked to expected outputs of the intervention (akin to a theory of change). In the lack of such a theory, the qualitative data gathered may be difficult to coherently analyze. That said, if there is a plan for doing this in another way, authors could include that.\n\nData analysis\nThere is limited information on how quantitative and qualitative data will be analyzed. This will have implications on the data collection methods and sampling details proposed. Also, see above about evaluation component and (lack of) details of the analysis proposed.\n\nIs the rationale for, and objectives of, the study clearly described? Partly\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "6442", "date": "29 Mar 2021", "name": "Anand Ahankari", "role": "Author Response", "response": "Response to comments from reviewer 1 (Prashanth N Srinivas) 1. The paper is a study protocol of research proposed in Osmanabad district of Maharashtra, India to improve evidence-base for understanding and acting upon gender-based violence (GBV). The study seeks to understand gender equality related knowledge, attitudes and practices among adolescents on one hand, and evaluate an ongoing intervention to reduce gender-based violence, called “Responsible Couples” (RC). The authors make a strong case for the need for such evidence (from adolescents, where interventions targeting the problem ought to begin) as well as the relative lack of effectiveness evidence from interventions to reduce GBV. Authors indicate that this could be the first of its kind large survey on this topic in Maharashtra, and perhaps among Indian adolescents. In addition, their remarks on the research capacity-building and academic exchange involved are also noted with appreciation. Response: Thank you for your careful reading of the manuscript and positive feedback. We believe your comments and suggestions have helped us to improve the quality of the manuscript. Please find below our point-by-point responses and revisions. 2. Background The background provides a published estimate of domestic violence (DV) ever and DV in the past 6 months. The study cited surveyed households in one city of south India, whereas the background states this as being an estimate for south India, which may not be an appropriate representation of the original study’s findings. Response: We have revised the introduction/background as follows. A recent study involving women from marginalised groups in Bangalore city based in southern India reported that over 50% of women had experienced physical domestic violence ever, and 27% faced physical violence in the past six months 2 . 3. Improving coherence through more contextual information and clarifying objectives A project goal is separately provided in addition to the research objectives. The two research objectives focus on (1) survey tool development for adolescents, and (2) evaluation of the RC program. The project goal is quite broad and both objectives fit within this goal, but it is unclear how these two objectives come together. Is there any specific reason these two objectives have been chosen? What was the rationale for choosing this particular intervention? Perhaps this is related to specific contextual information that may not be part of the protocol. Researchers could consider a section that describes the particular setting where the study is being planned as well as perhaps a section that describes work leading up to the study to provide the reader an understanding of the logic/reasoning in choosing these two objectives in order to work towards the project goal. Response: We have provided a more detailed explanation. DEVELOP project is designed primarily to support new research collaborations by generating evidence mainly from rural areas of the Maharashtra state of India. The evidence generation is proposed by two key objectives which fit under the overarching project goal as explained below. Research work will involve local staff working full-time at HMF (NGO partner) who will be trained to develop their research capacities and will be engaged in data collection work. Research undertaken through both objectives by a local team will provide them ‘real world’ experiences supporting their future career development. Secondly, our two key partners (SWISSAID and HMF) will benefit from such evidence to inform their on-going as well as forthcoming projects on gender equality in India. The first objective will help our key partners plan projects involving young people through their future expansion, and the second objective will contribute to improve their on-going intervention approach. SWISSAID works with several Indian NGOs, thus findings will be useful in other areas of Maharashtra and nationally in India to design gender equality related work. Research findings, capacity building initiative and collaboration work will also offer valuable experiences for all partners involved to plan future initiatives. The proposed structure helped design the project in line with our funding requirements where local capacity building and research goals are incorporated into this model. 4. While the background indicates that the authors will undertake a survey among adolescents (and indeed the data collection and subsequent activities confirm this, the first of the two objectives that include this appears to stop short at “…inform(ing) the development of a survey tool”. This gives an impression of this being a tool-development/feasibility study only. Clarify. Response: We have revised objectives to improve clarity and ensure that survey tool development and its proposed use is reflected with study location. To develop a survey tool and measure gender equality related knowledge, attitudes, and behaviours in Indian adolescents using this tool in rural populations of the Maharashtra state of India. To conduct a qualitative evaluation of the current DVA reduction intervention–the ‘Responsible Couples’ project implemented by HMF in rural areas of the Maharashtra state of India. 5. Selection and recruitment The project identifies 70 villages which correspond to the area of work of partner NGO as being the study area. It is unclear from the information provided how much of the district this covers, and what the original logic is for the partner NGO to choose these villages. Since a survey is being planned, the reporting of its findings will depend on the overall choice of study site and sampling and hence further information has to be provided to enable an understanding of the study area. Some information on the larger context in terms of how this district compares with other districts in the state with respect to GBV are useful (if available). An estimate of what proportion of the district is covered by these villages is also useful. Response: To the best of our knowledge, there are no published studies from this region to report, but we have provided information on the study area and connected this further with NGO’s work. The study area consists of 70 villages from HMF’s project field, located in the Osmanabad district of Maharashtra state of India. HMF accelerated its programmes in this region following the 1993 earthquake to support emergency relief activities. This geographic area is known for limited healthcare facilities, poor infrastructure, and is also among marginalised regions nationally. Therefore, HMF’s work focus has been in this region over 25 years where various welfare, education, health and development projects are regularly implemented. Majority of the population in the Osmanabad district is in rural areas (approximately 83% rural and 17% urban), and has about 733 villages in total across its 8 blocks. The proposed 70 villages are from two blocks namely Tuljapur and Lohara. The total district population is about 1.6 million and our study area has about 120-140,000 people across 70 villages. 6. Authors indicate that the age of eligible respondents for the survey shall be 16 to 19. How will this be verified (if at all)? Will this be based on self-reporting of age? Or will researchers have an estimate/list from prior work? Response: Data collection team will use self-reported age. All project staff were made aware about this requirement and such eligibility information is shared verbally as well as through participant information sheet. The study will be open to both boys and girls aged 16 to 19 only. Information on this age related eligibility will be shared through staff trainings including field level support personnel who will ensure that this is shared correctly with potential eligible participants. Self-reported age will be verified verbally on the day of data collection by members of the local research team. Age requirement is mentioned on the participant information sheet which is provided in advance to eligible candidates (Supplementary 1, Extended data) 7. Participants will be asked to report their age on the data collection form. 7. Will estimates/identity of adolescents in these villages be available to researchers from the existing work of partner NGO? If yes, specify how this data will be dealt with and used/not used. Authors may consider comparing their village-wise sample with adolescents in the village to come up with an estimate of their overall survey coverage among adolescents. Response: Such information is not collected, stored or used anytime during the project due to the ethical implications. At the planning stage of the project, we considered estimating the overall survey coverage, but this was not feasible because unfortunately information on the numbers and socio-demographic characteristics of adolescents by village or region was not available. 8. If adolescents who are unable to read/write turn up, what will the team do? There are both technical and ethical implications of this decision to exclude this group, even if the numbers in this group are small. More so given that educational status has been declared in the background to influence GBV. Response: Indeed, only those who are able to read and write will be able to participate in this study. This was preferred instead of interview questionnaire to ensure confidentiality and avoid reporting bias. From the experience of our partners and the lead researcher on our team who have previously conducted five research projects in this area, overall literacy skills among adolescents are sufficient to fully understand and participate in the survey. We have acknowledged this limitation in our revised draft. We acknowledge that those who may not be able to read and write will not be eligible to participate in our study. However, considering our ethics processes and confidentiality needs to report gender equality related response with necessary privacy, self-reported approach is preferred. 9. Sample size Authors state that one of the reasons for choosing a sample size of 1000 is “…to ensure that results from this study will be applicable to rural areas of Maharashtra state.”. This will need to be explained clearly. Specifically, the concern is with respect to (a) how would any sample size defined within a set of villages in one district of the state “…be applicable” to the entire state? Perhaps the authors seek to achieve analytical generalisability of their findings through mixed-method/qualitative inquiry and demonstrate the relative similarity of few/many/all other districts to this district with respect to the area of inquiry, but this cannot be based on a particular sample size in my assessment. It is also noted that another logic to arrive at the size has been the resource availability. On the contrary, could an effort at achieving a sampling strategy of smaller numbers of household visits be used to further minimize sample numbers if needed? I understand that the work may have already begun, in which case, authors need to clarify this section appropriately. Authors mention a stratified sampling approach, but the survey is being conducted by invitation to a “station” at each village. How will the 125 per each age-stratum and 500 from each gender be achieved if there is a patterning in age of children who turn up at the station (for instance, older boys being less “shy” of such a station, or for instance if some stations for reasons unknown are not acceptable/accessible for particular age/gender respondents). Given that the researchers do not identify specific strategies to ensure the sample across strata, this stratified sampling appears more to be a desire at ensuring distribution of sample across all age-groups rather than an actual sampling strategy? Clarify. Response: We have revised this section to improve clarity: The adolescent survey will be conducted to collect a minimum of 1000 questionnaires. This number is based on discussions with project partners to ensure that study is deliverable in given resources and time. Research findings may be applicable to wider areas of rural Maharashtra state. The proposed target of 1000 is set considering three months will be available to collect data, however if permitted higher sample size will be achieved depending on project progress. A similar strategy was used to conduct research in this field area involving adolescent girls 8. In order to collect representative data by age and gender, a stratified sampling effort will be undertaken. In total, a minimum 1000 questionnaires will be completed with an aim to collect 125 from each age group (16,17,18,19 years), and 500 from each gender. This is the ideal sample scenario; however, no participants will be turned away on a data collection day even if the said number has already been achieved. This strategy is proposed to work towards attaining similar numbers of participants across all age groups to conduct subgroup analysis, if permitted. Any limitations arising from our research design and data observations will be reported along with research findings. 10. RC program evaluation In terms of improving the coherence between these 2 objectives, and also given that both are being implemented in the same district, it is useful to understand if the 40 villages where RC will be evaluated are a sub-set of the 70 where the quantitative survey will be conducted? Response: Yes, the said 40 villages make the total of 70. We have revised the manuscript accordingly. The ‘Responsible Couples’ project is currently (in 2019) being implemented in 40 villages of Osmanabad district of Maharashtra state, India. These 40 villages are from the wider 70 village network outlined earlier where our NGO partner (HMF) is implementing development work. Each village has one local group comprised of 15 to 20 village members, who are voluntarily working towards gender equality in their community. 11. The RC group in each village: Are these volunteers or paid staff of the project? Clarify. Response: Village level gender equality group members work voluntarily. We have mentioned this in the revised text above. 12. Given that the objective is to evaluate an intervention which has multiple components, an overall evaluation approach to the intervention is missing. Partly, this could be dealt with by providing more details of how the qualitative data will be analyzed from the FGDs. Also to some extent, the evaluation will only engage with relative insiders (the village level RC group) and will not include perspectives of those who \"received\" the intervention, a limitation. Authors could consider including a clear schematic/narrative on how the intervention is expected to work outlining the various intervention inputs, assumptions being made and how it is linked to expected outputs of the intervention (akin to a theory of change). In the lack of such a theory, the qualitative data gathered may be difficult to coherently analyze. That said, if there is a plan for doing this in another way, authors could include that. Authors response: We have secured a follow-up funding where qualitative interviews were conducted through the DEVELOP Phase 2 work. This involved in-depth interviews with men and women who utilised project services. Research data collection for this was completed in 2020, and currently this data are being prepared for analysis. We have mentioned this in our evaluation approach which is a new section included in the protocol. ‘Responsible Couples’ project evaluation The proposed qualitative data collection through FGD with village level gender equality groups will contribute towards a full evaluation report. The proposed component is preferred during this initial stage of our work (DEVELOP Phase 1, 2018-19) to investigate community/village level change on gender equality related attitudes, behaviors to prevent and reduce DVA against women/girls. The future research (DEVELOP Phase 2, 2020-21) aims to conduct qualitative interviews with service users (men and women) who have used project services offered by village level groups and the NGO partner (HMF). For such future expansion, an independent funding will be sought. Qualitative data from both, service providers and users along with project monitoring reports by our partner organizations will generate evidence towards the final evaluation findings. We acknowledge that evaluating project services only from providers perspective will not be sufficient, thus future initiatives are planned, however such details are not included in this protocol considering it is beyond the current project’s funding and timeframe (DEVELOP Phase 1, 2018-19). 13. Data analysis There is limited information on how quantitative and qualitative data will be analyzed. This will have implications on the data collection methods and sampling details proposed. Also, see above about evaluation component and (lack of) details of the analysis proposed. Authors response: We have revised data analysis plan for both qualitative and quantitative sections as follows. For quantitative component Incomplete questionnaires will be discarded from analysis and stored for auditing purposes. Details on such process including data validation steps will be provided in study methods/results. Survey responses will be analysed in Stata (StataCorp, College Station, Texas, USA) and/or SPSS (IBM) using descriptive statistics, tests of statistical significance, and reliability coefficients. Summary of all collected data will be presented through frequency and percentages for findings from the gender equality tool and research participant demographics. Cronbach’s alpha score for the gender equality scale will be provided. The gender equality tool will be used to calculate a total score for each participant, and will be also used to report overall observations from on our study population. This score will be used as a continuous outcome of interest for linear regression purposes. In such analysis, data on individual sociodemographic parameters will be used as an independent exposure variables. Regression analysis will be adjusted depending on availability of data, statistical guidance and published evidence. If data permit, then additional analysis such as logistic regression will be conducted along with comparing results across villages/blocks. Results will be reported in line with STROBE guidelines 11 , and will be submitted for a peer review publication. For qualitative component Qualitative data from the focus group discussions will be analysed thematically, where two researchers will independently code data and synthesise findings into themes. This will be informed by Braun and Clark’s deductive reasoning methodology 12. Additional inputs will be provided by senior qualitative researchers to supervise this process and will also contribute towards such analysis. Furthermore, researchers working on this data will then meet to discuss areas of agreement and disagreement and reach consensus on the coding tree, illustrative quotations, and interpretation. All data findings will be shared with key project partners and data collection to conduct internal peer reviews and checks prior to finalising themes and key findings. Presentation guidelines such as COREQ will be followed wherever deemed necessary 13 . All agreed themes and sub-themes will be reported in the study results." } ] }, { "id": "77963", "date": "26 Feb 2021", "name": "Katharina Goessmann", "expertise": [ "Reviewer Expertise partner violence", "violence against women", "psychotraumatology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript, entitled “aDolescents gEnder surVey, rEsponsible coupLes evaluatiOn, and capacity building Project in India (DEVELOP): a study protocol“, presents a study protocol of a proposed study to be conducted within a capacity building project (called DEVELOP) that seeks to plan and perform programs to address gender-based violence (GBV) in India, which is a serious issue with numerous negative consequences, as the authors outline in their introduction. The manuscript reports on two components of the project: a quantitative survey on gender-related knowledge, attitudes and practices among adolescents to improve the evidence base in this regard, as well as qualitative evaluation of an ongoing GBV intervention (“Responsible Couples”, RC) in Osmanabad district of Maharashtra, India. In addition to those points raised by the first reviewer, I have a few remarks that might help to further improve the manuscript.\nIntroduction:\nFrom the Title, Abstract and the first part of the introduction, it is not clear whether the study focuses on domestic/partner violence against women, or refers to any kind of GBV against women. Please clarify this from the beginning; also, in the summary of literature from studies reporting GBV globally and in India.\n\nThe authors state that existing intervention programs to reduce GBV have predominantly focused on women. I suggest adding some more information here on why it is important to include men/boys in those efforts.\n\nIt would also be interesting to learn a bit more about the background of the DEVELOP project already in the introduction, such as what are its expected advantages, whether there are other comparable approaches and so on.\n\nQuantitative component:\nSelection and recruitment:\n\nThe envisaged number of participants in the quantitative survey is 1000. What is the time frame in which you are planning to achieve this number?\n\nHow will the under-age participants give their informed consent? Is the informed consent of parents/guardians required here?\n\nSurvey questionnaire (Extended data): In my opinion, the wording of some items could be slightly changed in order to avoid misunderstandings and allow meaningful interpretation:\nPart A, item 5: In its current wording, the item might be understood as asking for the status quo of what “a women’s role” usually is in society or what the adolescents experience in their daily life. Like this, their answer to the item is not necessarily reflective of a lack of support for gender inequality, as it does not directly ask whether the respondent agrees with this widespread societal role of women, or if they think it should be like that. If the item aims to assess the adolescents’ gender beliefs and attitudes, I suggest rephrasing it slightly, for example like this: A woman’s role should be taking care of her house and family members / A real/good woman’s role is taking care of her house and family members. Similarly, in item 8.\n\nPart B, item 10: In this item to it is unclear whether it is asking for the adolescents’ opinion/belief in this regard, or for their experience. This should be either rephrased or carefully considered in the interpretation of the data.\n\nQualitative component: Data collection:\nGiven the sensitivity of the topic, the participants’ gender identities should be considered. Will the focus groups consist of members of all genders or will they be separate for men and women members of the local gender promotion groups? Or are they all female anyway? And what about the gender of the research staff conducting the focus groups?\n\nConclusions:\nThe authors state that “the project will provide opportunities to train and engage over 10 researchers and practitioners in Maharashtra state of India to improve their knowledge, develop new research skills, and enhance their experience of collaborating with international partners“. From the described procedures, this expected outcome is not apparent. Please elaborate on what this assumption/aim is based on.\n\nMinor corrections:\nFigure 1: the second lowest box on the right should read “Qualitative component”\n\nIs the rationale for, and objectives of, the study clearly described? Partly\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "6443", "date": "29 Mar 2021", "name": "Anand Ahankari", "role": "Author Response", "response": "Response to comments from reviewer 2 (Katharina Goessmann) 1. The manuscript, entitled “aDolescents gEnder surVey, rEsponsible coupLes evaluatiOn, and capacity building Project in India (DEVELOP): a study protocol“, presents a study protocol of a proposed study to be conducted within a capacity building project (called DEVELOP) that seeks to plan and perform programs to address gender-based violence (GBV) in India, which is a serious issue with numerous negative consequences, as the authors outline in their introduction. The manuscript reports on two components of the project: a quantitative survey on gender-related knowledge, attitudes and practices among adolescents to improve the evidence base in this regard, as well as qualitative evaluation of an ongoing GBV intervention (“Responsible Couples”, RC) in Osmanabad district of Maharashtra, India. In addition to those points raised by the first reviewer, I have a few remarks that might help to further improve the manuscript. Response: Thank you for your careful reading of the manuscript and positive feedback. We believe your comments and suggestions have helped us to improve the quality of the manuscript. Please find below our point-by-point responses and revisions. 2. Introduction: From the Title, Abstract and the first part of the introduction, it is not clear whether the study focuses on domestic/partner violence against women, or refers to any kind of GBV against women. Please clarify this from the beginning; also, in the summary of literature from studies reporting GBV globally and in India. Response: We have now revised this across the study protocol by using two key terms- Domestic Violence and Assault (DVA), where intimate partner violence (IPV) is the most common form. Revisions were made to ensure consistency across our writing. 3. The authors state that existing intervention programs to reduce GBV have predominantly focused on women. I suggest adding some more information here on why it is important to include men/boys in those efforts. Response: We have now expanded further on this section as follows. Although the need to reduce DVA and empower women in India is widely recognised, there is very limited information available on gender equality-related knowledge, attitudes, and behaviours among Indian adolescents, who are an important age group to target with specific interventions. Interventions to reduce DVA were primarily focused on women for a long-time, and men were not involved in such intervention initiatives. This results in a lack of awareness in men who are often perpetrators subjecting risks to interventions targeted to reduce DVA. However, creating such awareness among men is challenging as it requires change in their attitudes, behaviours by empowering them with knowledge on the importance of gender equality and also on negative impacts of DVA on women and children. Development work to initiate such change requires challenging existing social norms, which come from centuries-old cultural practices, where discrimination against women/girls caused gender-based allocation of resources, work and opportunities across lifespan. Therefore, involvement of men in gender equality related work broadly aims to initiate and sustain a change at individual, family and community level to improve health and wellbeing of women. There is very limited research on the effectiveness of DVA reduction interventions especially from rural and difficult to access communities. In India, men engagement expanded in the recent decade thus evaluating such programmes are imperative to inform future work. 4. It would also be interesting to learn a bit more about the background of the DEVELOP project already in the introduction, such as what are its expected advantages, whether there are other comparable approaches and so on. Response: Details of the conceptualisation of the DEVELOP project, its relevance and collaborations are explained at the start of the protocol prior to goals and objectives. DEVELOP project is designed primarily to support new research collaborations by generating evidence mainly from rural areas of the Maharashtra state of India. The evidence generation is proposed by two key objectives which fit under the overarching project goal as explained below. Research work will involve local staff working full-time at HMF (NGO partner) who will be trained to develop their research capacities and will be engaged in data collection work. Research undertaken through both objectives by a local team will provide them ‘real world’ experiences supporting their future career development. Secondly, our two key partners (SWISSAID and HMF) will benefit from such evidence to inform their on-going as well as forthcoming projects on gender equality in India. The first objective will help our key partners plan projects involving young people through their future expansion, and the second objective will contribute to improve their on-going intervention approach. SWISSAID works with several Indian NGOs, thus findings will be useful in other areas of Maharashtra and nationally in India to design gender equality related work. Research findings, capacity building initiative and collaboration work will also offer valuable experiences for all partners involved to plan future initiatives. The proposed structure helped design the project in line with our funding requirements where local capacity building and research goals are incorporated into this model. 5. Quantitative component: Selection and recruitment: The envisaged number of participants in the quantitative survey is 1000. What is the time frame in which you are planning to achieve this number? Response: We have revised these details as mentioned earlier in this letter in response to the first reviewer and also addressed your question. The revision is outlined below. The adolescent survey will be conducted to collect a minimum of 1000 questionnaires. This number is based on discussions with project partners to ensure that study is deliverable in given resources and time. Research findings may be applicable to wider areas of rural Maharashtra state. The proposed target of 1000 is set considering three months will be available to collect data, however if permitted higher sample size will be achieved depending on project progress. A similar strategy was used to conduct research in this field area involving adolescent girls 8. In order to collect representative data by age and gender, a stratified sampling effort will be undertaken. In total, a minimum 1000 questionnaires will be completed with an aim to collect 125 from each age group (16,17,18,19 years), and 500 from each gender. This is the ideal sample scenario; however, no participants will be turned away on a data collection day even if the said number has already been achieved. This strategy is proposed to work towards attaining similar numbers of participants across all age groups to conduct subgroup analysis, if permitted. Any limitations arising from our research design and data observations will be reported along with research findings. 6. How will the under-age participants give their informed consent? Is the informed consent of parents/guardians required here? Response: Considering our ethics approval, adolescents over 16 years were permitted to provide consent for their own participation. Furthermore, bearing in mind local ethics requirements in India, eligible participant were verbally informed about the project and were also given participant information sheet to take home and decide on their participation following discussions with their local guardian/parents. There was no financial incentive provided to avoid any possible coercion. No personal identifiable information was required or ever collected from participants where self-reported questionnaire strategy was used. 7. Survey questionnaire (Extended data): In my opinion, the wording of some items could be slightly changed in order to avoid misunderstandings and allow meaningful interpretation: Part A, item 5: In its current wording, the item might be understood as asking for the status quo of what “a women’s role” usually is in society or what the adolescents experience in their daily life. Like this, their answer to the item is not necessarily reflective of a lack of support for gender inequality, as it does not directly ask whether the respondent agrees with this widespread societal role of women, or if they think it should be like that. If the item aims to assess the adolescents’ gender beliefs and attitudes, I suggest rephrasing it slightly, for example like this: A woman’s role should be taking care of her house and family members / A real/good woman’s role is taking care of her house and family members. Similarly, in item 8 Part B, item 10: In this item to it is unclear whether it is asking for the adolescents’ opinion/belief in this regard, or for their experience. This should be either rephrased or carefully considered in the interpretation of the data. Response: We prepared questions based on published literature on this subject, followed by discussions among team members, and then piloted involving adolescents from our study region. This process helped us identify issues around how certain questions were framed and those were revised. Further, advice on providing examples on some questions was implemented carefully. Nevertheless, we acknowledge that the survey tool could be further improved as you have explained. Due to the timeline of our project, data collection and analysis activities are now completed. However, we highly appreciate your advice, which is certainly very useful during future development of our survey tool. We have noted your guidance carefully to make further improvements and then aim to again pilot our survey tool in both rural and urban populations in India for future projects. 8. Qualitative component: Data collection: Given the sensitivity of the topic, the participants’ gender identities should be considered. Will the focus groups consist of members of all genders or will they be separate for men and women members of the local gender promotion groups? Or are they all female anyway? And what about the gender of the research staff conducting the focus groups? Response: This is indeed a very important area where project team discussed in detail during research design. All men and women work together through village level gender equality groups, and were also trained jointly as a team by the NGO partner over two years of period. Thus, all FGD will involve men and women during discussions. Two qualitative researchers collected these data having a combination of 1 male and 1 female researcher. As this was possible following a successful recruitment only, thus was not mentioned in this protocol. Nonetheless, we have now included following information to state this intention. Focus group discussions will be conducted, transcribed, and translated by two experienced facilitators with bilingual skills (Marathi and English) under the supervision of the research team. Efforts will be made to recruit one male and one female researcher for this data collection task to achieve a gender balanced approach. This will provide a comfortable environment for all men and women members/study participants. Further, both researchers will lead on 6 FGD sessions each to provide equal opportunities for skill development through peer and supervisory feedback in line with our capacity development objectives. 9. Conclusions: The authors state that “the project will provide opportunities to train and engage over 10 researchers and practitioners in Maharashtra state of India to improve their knowledge, develop new research skills, and enhance their experience of collaborating with international partners“. From the described procedures, this expected outcome is not apparent. Please elaborate on what this assumption/aim is based on. Response: We have revised this information and also linked with the project development and goal where capacity building initiative was mentioned. The revised conclusion is as follows. The DEVELOP project will contribute to research capacity building and evidence-based practices in a resource limited setting to achieve our overarching project goal. The project will provide opportunities to train and engage a team of 12 local staff includes data collectors, assistants, researchers in the Maharashtra state of India to improve their knowledge, develop research skills, and enhance experiences of all institutes on international collaborations. It is expected that the project will help partners involved from India and the UK to continue research and also development work on the adoption, implementation, and scale-up of evidence-based gender equality interventions in Maharashtra and other Indian states and territories. To the best of our knowledge, this will be the first survey from the Maharashtra state of India, and one of the largest surveys, measuring gender equality-related knowledge, attitudes, and behaviours among rural Indian adolescents. The survey findings will generate new valuable insights into how adolescent groups could be engaged in the future to improve gender equality in Indian communities. The qualitative evaluation will inform the implementation of the ‘Responsible Couples’ intervention and strategies to improve the same through future expansion. It will also have policy implications for HMF, SWISSAID, and other organisations seeking to reduce DVA and empower women in Maharashtra and other parts of the country. Considering diverse Indian culture, practices, and beliefs, the study results should be interpreted carefully beyond the population studied. Minor corrections: 10. Figure 1: the second lowest box on the right should read “Qualitative component” Response: Thank you for noting this error. We have now requested to change this to ‘Qualitative’. A figure indicating this change is also attached with the revised submission and submitted to the F1000." } ] } ]	1	https://f1000research.com/articles/8-958
https://f1000research.com/articles/10-245/v1	26 Mar 21	{ "type": "Opinion Article", "title": "Proposal of a novel Artificial Intelligence Distribution Service platform for healthcare", "authors": [ "Antti Väänänen", "Keijo Haataja", "Katri Vehviläinen-Julkunen", "Pekka Toivanen", "Keijo Haataja", "Katri Vehviläinen-Julkunen", "Pekka Toivanen" ], "abstract": "In this paper, we focus on presenting a novel AI-based service platform proposal called AIDI (Artificial Intelligence Distribution Interface for healthcare). AIDI proposal is based on our earlier research work in which we evaluated AI-based healthcare services which have been used successfully in practice among healthcare service providers. We have also used our systematic review about AI-based healthcare services benefits in various healthcare sectors. This novel AIDI proposal contains services for health assessment, healthcare evaluation, and cognitive assistant which can be used by researchers, healthcare service provides, clinicians, and consumers. AIDI integrates multiple health databases and data lakes with AI service providers and open access AI algorithms. It also gives healthcare service providers open access to state-of-the-art AI-based diagnosis and analysis services. This paper provides a description of AIDI platform, how it could be developed, what can become obstacles in the development, and how the platform can provide benefits to healthcare when it will be operational in the future.", "keywords": [ "Artificial Intelligence", "AI Platform", "Healthcare AI", "AI Distribution" ], "content": "1 Introduction\n\nImplementing Artificial Intelligence (AI) methods in healthcare services is the key element for reducing healthcare costs and improving health outcome. This have been proven in several studies where benefits of AI methods have been compared with the traditional process1. Even if there are proven benefits when implementing methods using AI into healthcare services there are also many issues which need to be taken into account if we would like to see AI to have a breakthrough in the field of healthcare. We need to proceed with AI as one technology of augmented intelligence solution where AI will be helping clinicians and healthcare decision makers providing more accurate diagnosis, predictions, and guidance. We can accept AI as a natural evolution in healthcare information technology (IT) only when all actors can see benefits of AI and these benefits can be identified and approved by all actors in healthcare processes. To provide better chance of success to AI evolution or even “revolution”, we must provide the possibility to healthcare services developers and researchers to access scientifically studied and validated state-of-the-art AI methods and health information databases to enhance healthcare processes and healthcare outcome. These AI methods and databases should be accessible transparently and without high costs or regulatory barriers. Some companies, such as GE Healthcare, are already working with AI platforms which make quick implementation of proprietary AI algorithms or third-party AI algorithms possible inside one platform2.\n\nOur novel AIDI platform will be designed to provide easy access interface for healthcare service providers, healthcare IT companies, and researchers for utilizing state-of-the-art third party or self-developed AI methods for specific healthcare use cases. It contains cloud-based AI services which can be integrated to other end user organizations’ healthcare service platforms with open application programming interfaces (APIs). These AI services can be used by clinicians or other healthcare stakeholders for providing support in decision making. AIDI can be used for new AI methods validation and evaluation purposes. The goal of the state-of-the-art AIDI platform is to provide more accurate and cost-effective diagnosis, predictions to healthcare, and give researchers and developers a platform to validate and evaluate new AI methods.\n\nThe paper is organized as follows. Section 1 provides an overview of the AI benefits in healthcare sector and rationale for the development of AIDI platform. Section 2 gives an architectural and technical overview of AIDI platform. Section 3 discusses use cases, benefits, and issues of AIDI platform development. Finally, Section 4 concludes the paper and sketches some future work.\n\n\n2 AIDI architecture and technical overview\n\nIn this section we provide an abstract architectural design of AIDI and details of its interfaces, application/software stack, and intended use. Figure 1 shows the basic architecture of AIDI containing actual server with data repository interfaces for data repositories and AI service providers, proprietary and public AI methods and algorithms database, and interface for end users. AIDI services will be operated in a cloud environment such as Microsoft Azure or similar.\n\nAIDI contains applications, service models, and data interfaces to end users, such as healthcare service development companies and healthcare service providers, researchers, and individuals. AIDI also has data interfaces to health databases, imaging databases, and AI service and algorithm providers. Figure 2 shows AIDI technology stack components.\n\nAIDI technology is capable of parallel processing of vast amounts of data from multiple data sources and will provide real-time analytics for research purposes and clinical decision making. Technology stack modules will be developed by using basic software development tools and open source software modules. It contains four layers: Front-End, Data Processing, Integration, and Data Storage.\n\nFront-end layer consists of web-based user interface (UI) for main application features, such as graphic tools and views of services and applications, selection of databases, analytics tools, and internal or third-party AI methods. Front-end contains also a separate analytics view where end users can see analytics of processed data and implement analysis view to own healthcare services or applications. It can be used by developers and researchers for own AI method testing and evaluation and comparison to other similar AI methods. Moreover, front-end contains UI for integration purposes where end users can configure own data integration settings, provide input data for AI, and implement own AI methods and data repositories. Front-end can be developed with Python and/or with ReactJS and data visualization can be enriched with streaming services visualization tools.\n\nData processing layer includes processing and analyzing methods for training data and user data with AI based processes. Pseudonymization and harmonization provide extra security layer to anonymization and harmonize data which can be received from multiple open or proprietary data lakes or data repositories. Machine learning processor can be used to perform data processing tasks, such as deep learning and supervised/unsupervised learning. Machine vision processor will provide AI, for example, image recognition and feature extraction needs. Natural language processor runs AI methods for language processing tasks, such as content extraction, classification, text generation, and machine translation. For data analysis and processing we can use third-party machine learning platforms, such as OpenAI3, Tensor Flow4, IBM Watson5, and Azure Cognitive Services6, and technologies, such as Python, and AI libraries, such as OpenCV, SciKit Learn, and Pandas.\n\nIntegration layer contains APIs for external databases, such as radiology imaging services (DICOM standard), hospital information systems (HL7 standard), and other third-party databases and data lakes (XML and JSON messaging formats). External data can be used as teaching data for AI methods. AI services API will provide integration to third-party AI algorithms and services, such as OpenAI, Tensor Flow, IBM Watson, and Azure Cognitive Services. AIDI open API will provide integration technology to end users to upload own data and AI methods to AIDI. For integration and data streaming AIDI can utilize streaming service platforms, such as Google Cloud Dataflow, Apache Kafka, or Amazon Kinesis.\n\nData storage layer has repositories for public and proprietary AI algorithms as well as database for AI teaching data retrieved from external databases or from end user database. End user database is used for maintaining users and organizations in AIDI services. AIDI has also service to import/export virtual machines and databases that AIDI can be easily installed to a cloud service, such as Microsoft Azure or to local server and database if AIDI would be used as proprietary service for specific company or organization use cases.\n\n\n3 Use cases\n\nWe propose modular AIDI deep learning solution with AIDI’s own AI methods and third-party AI methods. As a starting point AIDI will provide AI methods for use cases which are used in prediction, diagnostic, and analytics. Robotic surgery and surgery live monitoring use cases are excluded due to the critical life-threatening nature of these use cases. AIDI solution will be able to analyze high volume data and distributed data and provide diagnosis or analysis from input data in real-time. Catalogue of AI methods can be expanded based on use cases arising from healthcare service providers or healthcare information system developers. We have evaluated that AIDI can include AI services which are explained in sections 3.1-3.9.\n\nEfficiency of AI has been identified in eye diseases7, breast cancer8,9, and skin cancer10 diagnosis when using data mining, natural language processing, and machine vision methods. Furthermore, glucose level prediction is one of the use cases where AI methods have been utilized successfully11. AI methods can be used for tuberculosis diagnostics12 and prediction of psychosis13. AIDI platform can implement these studied AI methods to provide direct access to utilize these methods for customers and end users.\n\nPersonalised medicine is tailoring of medical treatment to the individual characteristics of each patient. The approach relies on the understanding of how a person's unique molecular and genetic profile makes them susceptible to certain diseases. Personalised medicine also focuses to provide detection of the diseases at an earlier stage when treatment activities can be applied more effectively14. AIDI can monitor patient diagnosis and data and based on data provided to pre-trained AI methods AIDI can suggest most effective treatments and/or medication to patient.\n\nAI methods to detect medication errors and increase medication adherence are used in healthcare sector. AI can be used to create screening system to detect potential medication errors and generate alerts to clinical decision support (CDS) systems and electronic medical record (EMR) systems15. Machine vision and neural networks have been used efficiently to monitor medication intake by patients to enhance medication adherence. AIDI can provide access to these methods as a service to be utilized by healthcare service providers.\n\nIn clinical trials it is essential to identify potentially eligible patients to create more comprehensive clinical trials with lower costs. AI can be used to help in clinical trial design and to find patterns in large datasets for identifying most suitable patients before clinical trials start and AI can be used also for enabling more effective and accurate monitoring of patients during clinical trials. By using AI methods increase in enrolment in clinical trials has been observed. Furthermore, utilization of AI methods increases identification of eligible patients for clinical trials16.\n\nOn-going 2020 Covid-19 pandemic has increased the need to predict the progression of epidemics and pandemics. AI methods can be used for different epidemics by combining and analysing information of spatial spreading and behaviour data of the epidemic. As a result, estimation and prediction of epidemic spreading can be achieved17. Real-time forecasting of epidemic outbreak with AI have been used in COVID-19 forecasting in China with high accuracy18. Furthermore, AI methods can be used in managing on-going epidemic outbreaks by providing methods for identification of on-going epidemic outbreak, diagnosing and prognosing infections, and identifying possible therapeutic options19. AIDI methods can provide end users the solutions which utilize data from public and private health databases and data lakes to predict possibility for epidemics.\n\nAI has been used in connection with medical imaging for a long time and the use of AI methods has been extensively studied and proven to surpass human diagnosis in practical clinical work. In the field of medical imaging the AI technologies, such as DNN and DL, can produce remarkable improvement in healthcare outcome and they have proven to provide enhancement in speed, accuracy, and cost reduction in interpretation of medical images20. Medical image diagnostics can be utilized in healthcare for multiple cases, such as for identifying cardiovascular abnormalities, detecting common eye diseases by optical coherence tomography, detecting musculoskeletal injuries, identifying neurological diseases, identifying thoracic complications, and in oncology for screening common cancers21‐23. AIDI can provide these validated methods to end users for instant image analysis. Our plan is to provide AI methods for medical image diagnostics as among the first of AIDI services.\n\nAI utilization in health monitoring and preventive health consist of solutions for continuous health status monitoring, healthcare assessment tools, and symptom checking solutions which can be used by patients and healthcare professionals periodically or continuously. AI is also commonly used in personal health monitoring and giving personalized suggestions for preventive health based on user parameters, such as activity tracking, nutrition, sleep analysis, weight, body composition, and vital signs, such as blood pressure and blood sugar. Personal health apps are the most common health monitoring solutions which collect data from user smartwatch and smartphone and utilize AI to provide analysis and suggestions for end users. There are also clinical remote monitoring solutions which utilize AI, such as cardiology monitoring, sleep monitoring, and posture monitoring24,25. AIDI can provide periodical/intermittent analytics from the collected data and provide analysis to healthcare services development companies or research organizations.\n\nHealthcare services utilize AI based virtual nursing assistants in various use cases. By using virtual nursing assistants, hospitals will be able to reduce sudden hospital visits and reduce the load of healthcare professionals. Virtual assistant applications can listen, talk, and give advice/recommendations based on patient conversation, health history and health data of the patient. Common virtual nursing assistant solutions provide pre-diagnosis for patients and healthcare professionals based on the healthcare assessment questionnaire and patient history data before entering the primary care26,27.\n\nAIDI can be used to validate usability of new AI methods. AIDI will contain a growing number of AI methods for healthcare use cases. AIDI also provides access to training data and actual data. New AI methods can be evaluated and validated by comparing new AI method outcome (i.e., identification of accuracy, specificity, or sensitivity) and compare results with previous AI methods results. With this evaluation and validation service third-party AI method developers can create better AI methods which can be utilized in AIDI platform by companies or research organizations. Evaluation can be done by a specific multidisciplinary team and by scoring system in AIDI. This scoring provides end users valuable information about usability of selected AI method in specific use case.\n\nAs an example of AIDI we present the process flow of use case where clinicians want to analyse radiology image to identify possible lesion from the image. Steps are explained and shown in Figure 3:\n\n1. User sends radiology image to AIDI with information about use case (example case breast cancer analysis).\n\n2. Open API module analyses query and transmits information and asks for details from the user about the use case.\n\n3. AI services API select used AI service from data processing layer and select suitable algorithm from AI algorithm database or from third-party AI methods. In this example, selected service will be Machine Vision Processor and selected AI method is feature extraction.\n\n4. AIDI Machine Vision Processor continuously receives radiology images for diagnosis from imaging databases for AI algorithm training.\n\n5. Machine Vision processor uses training images for data pre-processing, classification, and feature detection.\n\n6. Training data for AI processor is received from RIS/PACS systems as background service and AI algorithms are continuously trained with collected data.\n\n7. Suitable AI algorithm for image analysis is selected and sent to Machine Vision Processor for real-time analysis.\n\n8. Results and findings from image analysis will be sent back to user and will be shown to user in Analytics UI. AI algorithm may also be altered during real-time analysis and sent as new enhanced version of the algorithm into the AIDI database.\n\n9. User will send diagnosis back to AIDI which can be used to improve the used AI algorithm accuracy. AIDI will monitor continuously the user-experienced diagnosis accuracy compared to diagnosis provided by AIDI.\n\nAdvantages for actors in healthcare\n\nWhen utilizing AIDI in healthcare use cases we can provide immediate benefits for all actors in care process and service development. For software and services development, there is less time used for training AI methods, since AIDI developers can use ready-made AI methods and modify methods and algorithms to meet their own use case specific needs. There is also no need to search for training data for AI algorithms, because AIDI contains interface for training data repositories. For healthcare service providers and caregivers, there is instant prediction, analysis, or pre-diagnosis possibility with the software which utilize AIDI platform features. AIDI can provide enhancement in diagnosis accuracy and diagnosis speed. For patients, healthcare customers, and people who are tracking their wellbeing, AIDI provides accurate and instant diagnosis, prognosis, and predictions based on personal health data and verified and trained AI analytics from large datasets.\n\nThe savings potential when using AI in healthcare is very high. In USA annual savings are expected to be 150 billion USD by 202628. When comparing this savings potential to total US healthcare expenditure in 2018 (3600 billion USD with 4,6% annual growth) we expect that US healthcare expenditure will be 5150 billion USD in 202629. With these values we evaluate that AI utilization can bring 2,9% reduction in healthcare expenditure in the US. We evaluate that same cost reduction percentage can be achieved also globally.\n\nAIDI benefits can be presented by the situation where state-of-the-art AI methods are integrated into AIDI and these services are utilized in healthcare in full scale. Benefits can be introduced in values, such as improvement in care outcome, cost reduction in healthcare, quality-of-life improvement among patients, and reduction in workload among healthcare professionals30.\n\nFor evaluation of AIDI methods in healthcare we also suggest a first version of the weighted scoring system for AI benchmarking. Our novel scoring provides benefit assessment for evaluating the implementation of new AI method in certain healthcare use case. Output is informed as AIscore. Same tool with modified parameter information can be used to evaluate AI method benefits in other business sector use cases. Parameters of the equation are: Scientifically Proven Improvement compared to traditional method (SPI), Training Data Score (TDS), Cost Reduction (CR), Quality-of-Life improvement (QoL), and Workload Reduction (WR). Parameters and weights will be inserted by evaluation team. Evaluation team has researchers and professionals in a particular field of healthcare. Parameter values inform parameter impact, effect, positive outcome, or improvement in certain use case of AI method. For example, parameter SPI can be set by specificity and sensitivity of AI method. Furthermore, parameter TDS can be set by evaluation training data quality and amount of training data parameters for the specific healthcare use case. Parameters CR, QoL and WR will be scored by evaluation team. Each of the parameters will be using weighted coefficient (Wn) based on the importance of parameter for specific use case. There is also acceptance parameter (AP) expressing whether method can be used for specific use case or not. AIscore will be developed and evaluated in future AIDI research projects.\n\nBy using AIDI and its validation of AI methods by AIscore, we can provide centralized AI distribution and utilization platform where all actors can benefit from effective and evaluated AI services. AIDI platform can also contain sales channel for evaluated AI methods which can be easily adopted for healthcare industry purposes. AI method developers can provide methods through AIDI as “Google Play” style where AI will be openly available.\n\nConcerns of AIDI adoption in healthcare\n\nAlthough AI methods in AIDI can provide scientifically proven enhancement in diagnosis and predictions accuracy, we must keep in mind that AIDI will only provide information and guidance for healthcare decision makers. AIDI platform or AI methods overall are not yet allowed to provide direct diagnosis. Healthcare professionals will always be responsible for providing diagnosis and final decision. There are also challenges which can emerge when AIDI services are utilized in healthcare. These issues are market acceptance and end user acceptance. Possible reasons for these issues are arising from the lack of curated healthcare data. Moreover, previous studies show that there might be resistance from healthcare professionals and patients to trust AI based decisions and diagnosis. Furthermore, people think that there is less “human touch” in care process when AI takes a greater role in healthcare. Moreover, shortage of regulations in AI in healthcare and data privacy can cause challenges. All these issues and challenges should be considered in planning phase of the AIDI project to enable acceptance of AIDI services to all end user groups and to all regulatory authorities.\n\n\n4 Conclusion and future work\n\nBased on our own research work related to AI benefits in healthcare and promising results from other researches in healthcare AI we can conclude that AI is beneficial to all actors in healthcare sectors. This “revolution” of AI in healthcare has not gone unnoticed by healthcare services development companies. Technology companies are continuously developing and releasing new AI services and even service platforms. Some of the platforms have similarity in AI methods distribution to our AIDI proposal. There are technology giants, such as IBM, Google, Microsoft, General Electric, and Siemens investing and developing AI solutions. We have evaluated that the drivers of the growth in healthcare AI development are based on scientifically proven advantages in care outcome and savings in healthcare costs with AI30. These factors have created the very high market growth. Also, other factors driving the market growth are the increasing volume of healthcare data and growing complexities of datasets driving the need for AI, the intensifying need to reduce towering healthcare costs, improving computing power and declining hardware costs, growing number of cross-industry partnerships and collaborations, and rising imbalance between health workforce and patients driving the need for improved healthcare services. Furthermore, one major factor growing the AI market growth is the adoption of AI by multiple pharmaceutical and biotechnology companies across the world. Global healthcare AI market is estimated to grow from 4,9 billion USD (estimated market size in 2020) to 45,2 billion USD in 2026 giving 44,9% of compound annual growth rate for this 6-year period31.\n\nOur future research work will focus on continuing this research with AIDI platform development and its features evaluation with healthcare used cases. First AI services provided by AIDI will concentrate on image diagnostics. This decision is based on our findings during this research where we noticed that healthcare service providers need methods which can easily be implemented into medical imaging and image diagnostics services. AI-based imaging and machine vision methods are widely adopted and accepted by healthcare professionals and companies who develop image diagnostic solutions for healthcare. Furthermore, we will expand AI methods database as well as provide more integrations to training data and third-party AI service providers. We will also analyse business potential and healthcare organizations acceptance of AIDI platform based on the market feedback and implement suitable AI methods for AIDI platform based on market needs. Moreover, we will apply funding for a new scientific project to bring AIDI platform alive.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "References\n\nVäänänen A, Haataja K, Vehviläinen-Julkunen K, et al.: Survey to healthcare professionals on the practicality of AI services for healthcare. F1000Res 2020. Publisher Full Text\n\nEdison Open AI Orchestrator: Reference Source accessed 5.10.2020.\n\nOpenAI: Reference Source accessed 3.10.2020\n\nTensorFlow: Reference Source accessed 1.10.2020\n\nIBM Watson Heatlh: Reference Source accessed 1.10.2020\n\nAzure Cognitive Services: Reference Source accessed 1.10.2020\n\nDe Fauw J, Ledsam JR, Romera-Paredes B, et al.: Clinically applicable deep learning for diagnosis and referral in retinal disease. Nat Med 2018; 24(9): 1342. PubMed Abstract \| Publisher Full Text\n\nMcKinney SM, Sieniek M, Godbole V, et al.: International evaluation of an AI system for breast cancer screening. Nature 2020; 577(7788): 89–94. PubMed Abstract \| Publisher Full Text\n\nPatel TA, Puppala M, Ogunti RO, et al.: Correlating mammographic and pathologic findings in clinical decision support using natural language processing and data mining methods. Cancer 2017; 123(1): 114–121. PubMed Abstract \| Publisher Full Text\n\nEsteva A, Kuprel B, Novoa RA, et al.: Dermatologist-level classification of skin cancer with deep neural networks. Nature 2017; 542: 115–18. PubMed Abstract \| Publisher Full Text\n\nClinicalTrials.gov: Adult accuracy study of the Elite 3 Glucose Censor (E3).Reference Source Accessed 18.7.2020\n\nLakhani P, Sundaram B: Deep learning at chest radiography: automated classification of pulmonary tuberculosis by using convolutional neural networks. Radiology 2017; 284(2): 574–582. PubMed Abstract \| Publisher Full Text\n\nCorcoran CM, Carrillo F, Fernández-Slezak D, et al.: Prediction of psychosis across protocols and risk cohorts using automated language analysis. World Psychiatry 2018; 17(1): 67–75. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStratified, personalised or P4 medicine: a new direction for placing the patient at the centre of healthcare and health education.Academy of Medical Sciences; Reference Source Accessed 6.10.2020\n\nSchiff GD, Volk LA, Volodarskaya M, et al.: Screening for medication errors using an outlier detection system. J Am Med Inform Assoc 2017; 24(2): 281–287. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCalaprice-Whitty D, Galil K, Salloum W, et al.: Improving clinical trial participant prescreening with artificial intelligence (AI): a comparison of the results of AI-assisted vs standard methods in 3 oncology trials. Ther Innov Regul Sci 2020; 54(1): 69–74. PubMed Abstract \| Publisher Full Text\n\nZamiri A, Yazdi HS, Goli SA: Temporal and spatial monitoring and prediction of epidemic outbreaks. IEEE J Biomed Health Inform 2014; 19(2): 735–744. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHu Z, Ge Q, Jin L, et al.: Artificial intelligence forecasting of covid-19 in china. arXiv preprint arXiv 2020; 2002: 07112.\n\nBragazzi NL, Dai H, Damiani G, et al.: How Big Data and Artificial Intelligence Can Help Better Manage the COVID-19 Pandemic. Int J Environ Res Public Health 2020; 17(9): 3176. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu X, Faes L, Kale AU, et al.: A comparison of deep learning performance against health-care professionals in detecting diseases from medical imaging: a systematic review and meta-analysis. Lancet Digit Health 2019; 1(6): e271–e297. PubMed Abstract \| Publisher Full Text\n\nMcKinney SM, Sieniek M, Godbole V, et al.: International evaluation of an AI system for breast cancer screening. Nature 2020; 577(7788): 89–94. PubMed Abstract \| Publisher Full Text\n\nEsteva A, Kuprel B, Novoa RA, et al.: Dermatologist-level classification of skin cancer with deep neural networks. Nature 2017; 542: 115–18. PubMed Abstract \| Publisher Full Text\n\nDe Fauw J, Ledsam JR, Romera-Paredes B, et al.: Clinically applicable deep learning for diagnosis and referral in retinal disease. Nat Med 2018; 24(9): 1342. PubMed Abstract \| Publisher Full Text\n\nDigital Health, Samsung: Reference Source accessed 22.10.2020\n\nHartikainen S, Lipponen JA, Hiltunen P, et al.: Effectiveness of the Chest Strap Electrocardiogram to Detect Atrial Fibrillation. Am J Cardiol 2019; 123(10): 1643–1648. PubMed Abstract \| Publisher Full Text\n\nLaranjo L, Dunn AG, Tong HL, et al.: Conversational agents in healthcare: a systematic review. J Am Med Inform Assoc 2018; 25(9): 1248–1258. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChambers D, Cantrell AJ, Johnson M, et al.: Digital and online symptom checkers and health assessment/triage services for urgent health problems: systematic review. BMJ open 2019; 9(8): e027743. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nArtificial Intelligence: Healhtcare’s new nervous system. Accenture consulting 2017Reference Source Accessed 19.9.2020\n\nNational Health Expenditure Data, Department of Health and Human Services: 2020Reference Source Accessed 20.10.2020\n\nVäänänen A, Haataja K, Toivanen P: A Narrative Review on AI in Healthcare. F1000Res 2020.\n\nArtificial Intelligence in Healthcare Market, Markets and Markets.Reference Source Accessed 22.10.2020" }	[ { "id": "92571", "date": "09 Sep 2021", "name": "Marko Jäntti", "expertise": [ "Reviewer Expertise Computer science" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper aims at developing a service platform AIDI (Artificial Intelligence Distribution Interface for healthcare) and showing its main components. The study focuses on explaining the structure of the artifact that authors have designed. The main artifact is an AI-based service platform. The structure of the paper is clear and the topic is very up to date and interesting. However, there are several issues that could have been improved.The Authors can consider them as improvement ideas.\nIntroduction The study refers to earlier work but does not provide a clear reference to those studies. \"proposal is based on our earlier research work in which we evaluated AI-based healthcare services\". Related to the above mentioned comment, the authors could have positioned their paper to previous academic studies and results more clearly. What does it mean in terms of this paper? For example, the title of the paper refers to \"novel\" platform. Positioning could have helped authors to identify and communicate better the elements that actually are \"novel\".\nThere are existing case studies that utilize already cloud & AI services (https://customers.microsoft.com/en-us/story/1399290856490209110-region-hovedstaden-health-provider-azure-en-we) and there are academic studies that mention Azure AI (R. Singh and N. Sharma, \"Machine Learning based Medical Information Analysis, Estimations and Approximations over Present Health Research Domain). Therefore, 'novel' parts could have been discussed further.\n\nSection 2 The section 2 describes the basic architecture of AIDI and four layers: Front end, data processing, integration and data storage. From a software development point of view, the selection of these layers is obvious and clear, however, one should ask 1) why these elements were chosen to be part of the artifact and 2) how the artifact (and opinions) have been designed /constructed by using the method. Transparency of this design / construction process is crucially important.\nHow authors could have addressed this better? If authors continue using a systematic review (\"We have also used our systematic review about AI-based healthcare services\") , there are guidelines how to apply the methodology. See for example: https://libguides.library.curtin.edu.au/systematic-reviews. Authors are encouraged to look this when they prepare their next systematic reviews.\nSection 3 Use cases is from a reviewer's perspective the most mature and interesting part of the article focusing on usage scenarios of AIDI platform. Authors cite existing AI and Healthcare-related literature showing the use cases for utilizing AI in healthcare. Literature presented here provides strong support for using AI components for example in predicting and governing pandemic outbreaks and scanning large datasets for identifying & selecting patients for clinical trials.\n\nAre the conclusions (opinions) drawn balanced and justified on the basis of the presented arguments? Authors could have followed the survey methodology in a more transparent way (defining exclusion/inclusion criteria and showing clearlier where their conclusions are based on?).\nThere is certainly always place for improvement in papers but as a whole, AI-based healthcare platforms deserve more attention and this paper manages to contribute to the field by describing the architectural elements of AI-powered healthcare platforms as well as usage scenarios derived from the literature.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Partly", "responses": [] }, { "id": "97304", "date": "08 Nov 2021", "name": "Lorenzo Faggioni", "expertise": [ "Reviewer Expertise Radiology", "Imaging informatics", "Artificial Intelligence" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nInteresting, quite exhaustive manuscript aimed at presenting a novel AI-based service platform proposal called AIDI (Artificial Intelligence Distribution Interface for healthcare). I think that it could be further improved as follows:\nPlease avoid mentioning commercial names or companies, unless indispensable to properly describe the data presented (e.g. GE Healthcare in the Introduction, Microsoft Azur at the beginning of Section 2).\n\nThe use cases from 3.1 and 3.8 are well structured and give a brief but systematic account of the potential and actual uses of AIDI. However, I would strongly suggest expanding each of them by mentioning the main literature references related to each use case in a dedicated table (reporting article title, first author, publication date, use case topic and a short summary of the methods and main findings). This would add significant value to the manuscript.\n\nUse case 3.6 (Medical image diagnostics). A big contribution of AI in the field of medical imaging that should be mentioned is the possibility not only to detect disease conditions (e.g., cancer), but also to provide insights about patient outcome and eligibility to some given treatments or not (e.g., molecular target therapies). Please elaborate on this.\n\nThe paragraph on \"Concerns of AIDI adoption in healthcare\" is an important one, yet it should be supported by literature references (which are currently lacking at all). Some useful references on this topic include (but are not limited to) include the following: 1) Coppola F., et al, doi 10.3389/fpsyg.2021.710982; 2) Triberti S., et al, doi 10.3389/fpubh.2020.00117\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-245
https://f1000research.com/articles/9-1416/v1	07 Dec 20	{ "type": "Brief Report", "title": "A national survey of community rehabilitation service provision for people with long Covid in Scotland", "authors": [ "Edward Duncan", "Kay Cooper", "Julie Cowie", "Lyndsay Alexander", "Jacqui Morris", "Jenny Preston", "Kay Cooper", "Julie Cowie", "Lyndsay Alexander", "Jacqui Morris", "Jenny Preston" ], "abstract": "Background: Over 50 million cases of COVID-19 have been confirmed globally as of November 2020. Evidence is rapidly emerging on the epidemiology of COVID-19, and its impact on individuals and potential burden on health services and society. Between 10–35% of people with COVID-19 may experience post-acute long Covid. This currently equates to between 8,129 and 28,453 people in Scotland. Some of these people will require rehabilitation to support their recovery. Currently, we do not know how to optimally configure community rehabilitation services for people with long Covid. Methods: This national survey aimed to provide a detailed description of current community rehabilitation provision for people with long Covid in Scotland. We developed, piloted, and conducted a national electronic survey of current community rehabilitation service provision for people presenting with long Covid symptomatology. Our sample were the Allied Health Professions Directors of all 14 territorial NHS Health Boards in Scotland. Fixed response and narrative data were analysed descriptively. Results: Responses were received from all respondents (14/14), enabling a national picture to be gained. Almost all Health Boards (13/14) currently deliver rehabilitation for people with long Covid within pre-existing services. Fatigue (11/14) and respiratory conditions (9/14) were the two most common presenting problems of patients. Most long Covid community rehabilitation services are delivered through a combination of face-to-face and digital contact (13/14). Conclusions: Community rehabilitation for people with long Covid is an emerging reality. This survey provides a national picture of current community rehabilitation for people with long Covid. We do not know how community rehabilitation can be optimally delivered for this population. This is vital as community rehabilitation services were already under pressure prior to the emergence of COVID-19. Further research is urgently required to investigate the implementation, outcomes and cost-effectiveness of differing models of community rehabilitation for this patient population.", "keywords": [ "COVID-19", "long Covid", "community rehabilitation", "allied health professions", "survey" ], "content": "Introduction\n\nSince the initial emergency of COVID-19 in Wuhan, Hubei Province, China in December 2019, the disease has rapidly spread across the globe, with more than 50 million cases now confirmed globally (9 November 2020)1. Among the consequences of COVID-19 is the impact of long Covid, where individuals are left with debilitating symptoms after the initial acute phase of infection2. To date there is no agreed definition of what constitutes long Covid, however it has been proposed that it is when individuals have symptoms extending three weeks beyond onset (post-acute COVID-19) and 12 weeks beyond onset (chronic COVID-19)3. Long Covid does not only affect people who were severely ill, but also people with milder symptoms, and those who were not hospitalised4.\n\nIt is estimated that 10–35% of people infected with COVID-19 may experience post-acute long Covid3–5. Globally this equates to between five and 17.5 million people who may experience debilitating aftereffects of the infection. Within Scotland (as of 15 November 2020) 1,086,353 people have been tested for COVID-19 with 81,294 people testing positive. Using this data, we can estimate that between 8,129 and 28,453 people may have post-acute COVID-19, with around 812 people remaining significantly unwell at 12-weeks, commonly due to organ damage6. Consequently, long Covid has received considerable media attention in Scotland and beyond, with action groups calling for more support for symptom management7. As around 10% of people who experience mild COVID-19 may go on to experience long-term symptoms8, it is important that rehabilitation is accessible to those presenting in community settings as well as being discharged from hospital.\n\nReported symptoms of long Covid vary widely. They commonly include respiratory, cardiopulmonary, neurological, musculoskeletal and mental wellbeing sequelae, as well as fatigue and loss of taste and smell4,5,9. The presentation and severity of these symptoms are variable. Several people who have long Covid report a non-linear journey of recovery and describe their symptoms as moving around their body, such that as one symptom abates, another appears9.\n\nA currently unknown number of people with long Covid will require rehabilitation to support their recovery and increase their quality of life. As with other long-term conditions, rehabilitation for people with long Covid should be multidisciplinary, comprehensive, and tailored to individuals’ needs, in order to maximise function, quality of life and participation in society10. Rehabilitation for long Covid is in its infancy. We do not currently know how rehabilitation can be optimally delivered for people with long Covid. Findings from a recent living systematic review found most publications have been expert opinion about how rehabilitation for long Covid should be delivered, indicating that high-quality research is required11. Understanding how to optimally deliver long Covid community rehabilitation is vital, as rehabilitation services need to cope with additional demand while continuing to provide rehabilitation for other, often vulnerable, patient populations12.\n\nThis paper reports a recently conducted national survey of current community rehabilitation provision for people with long Covid. The aim of the survey was to provide a detailed description of current community rehabilitation provision for people with long Covid across Scotland. We believe it to be the first published national survey describing long Covid rehabilitation models of practice. The survey is the first step in a programme of research to investigate how community rehabilitation can be optimally delivered for people experiencing long Covid.\n\n\nMethods\n\nUsing the Jisc online survey tool we developed and conducted a national electronic survey for the Directors of Allied Health Professions of all 14 territorial NHS Health Boards in Scotland. The aim of the survey was to discover their current service provision for rehabilitation of people presenting with long Covid symptomatology in the community. The survey is reported in keeping with recommended reporting guidance for surveys13.\n\nAn initial draft survey was developed by the study authors. This incorporated fixed item and narrative response survey questions, informed by the TIDieR Intervention Description checklist14:\n\nHow? How is long Covid rehabilitation delivered in your board area?\n\nWhy? What are the main problems that patients require rehabilitation for?\n\nWhat is provided? Please describe the service as fully as you can.\n\nWho provides? What professional groups are involved in delivering long Covid rehabilitation in your board area?\n\nHow/where is it provided? How do patients access long Covid rehabilitation in your board area?\n\nWhen and How much? Can you describe the timing and duration of typical long Covid rehabilitation in your board area?\n\nWe conducted a small pilot of the initial survey content with the Scottish Government’s Professional Advisor for Rehabilitation, the National Clinical Lead for Digital Health and Care, the Allied Health Professions’ Improvement Advisor for Healthcare Improvement Scotland, and the director of services of Chest Heart and Stroke Scotland. Minor changes to the survey wording were made based on their feedback. A copy of the final questionnaire used in this study is available as extended data.\n\nHealthcare in Scotland is primarily delivered through NHS Scotland’s 14 territorial Health Boards. Each Health Board covers a separate region. Together they cover the entire Scottish population. They are responsible for the protection and improvement of the health of the people in their region and the delivery of healthcare services. Each Health Board has a Director of Allied Health Professions. We invited all 14 Directors of Allied Health Professions to participate in this survey via an emailed letter. To minimise the potential of attrition bias, the letter from the study authors containing a link to the survey was emailed by the Scottish Government’s Professional Adviser for Rehabilitation to each of the Directors.\n\nThe online survey was launched on 14 October 2020 and closed on 6 November 2020.\n\nFixed response item data (Questions 1,2,3,4,5,6,8a,9) and numeric and narrative response item data (Questions 3a, 4a, 5a, 6a, 7, 8, 8ai) were analysed descriptively.\n\nAs the study surveyed current practice it did not require research ethics approval by the NHS. Data was stored on password protected University servers in compliance with European Union General Data Protection Regulation (GDPR) standards of data protection and storage. The covering letter to potential participants explained the reasons for the survey and that their anonymised responses may be published. Informed consent to participate and for the publication of results was implied through their return of the study questionnaire.\n\n\nResults\n\nWe received responses from all 14 Directors of Allied Health Professions, enabling a national picture of community rehabilitation service delivery for people with long Covid to be gained. An anonymised copy of all survey response data is available as underlying data.\n\nAlmost all Health Boards (13/14) are currently delivering rehabilitation for people with long Covid within pre-existing services. One Health Board has developed a new service for people requiring long Covid rehabilitation, and another is currently developing a new service. Data on the numbers of patients who have received long Covid rehabilitation to date were not available from most respondents (12/14), indicating that routine rehabilitation data collection methods are not yet universally established. In services that were able to provide referral number data (2/14), one respondent (from a rural island locality) stated that they had received a referral for one patient in total, while the Health Board with a specialist long Covid service stated that they had received 95 referrals in eight weeks.\n\nRespondents reported that the main symptoms requiring rehabilitation interventions were fatigue (11/14), respiratory conditions (9/14), musculoskeletal conditions (6/14), mental health (5/14), and neurological impairments (4/14). One respondent stated that patients who were referred to their service experienced fatigue (86%), respiratory symptoms (67%), reduced mobility/exercise tolerance (60%), low mood, anxiety, depression (43%), cardiac symptoms (24%), sleep disturbance (24%), and weight management concerns (12%).\n\nRespondents did not describe the therapeutic content of long Covid rehabilitation in any detail, referring instead to the professions that were involved in delivery of the service (see below). One respondent described their service providing energy conservation advice and assessment of aids and adaptations. Another respondent described their service as providing fatigue management, confidence building, muscle strengthening, anxiety management, nutritional advice, breathing re-education, and activities to support individuals to regain function. Another said their service used a combination of pulmonary rehabilitation and community reablement. A final respondent described their service as providing individualised goal setting based on symptomatic presentation.\n\nCommunity rehabilitation service provision for people with long Covid is multidisciplinary. Almost all services (13/14) include occupational therapy and physiotherapy. Many include dietetics (11/14) and speech and language therapy (9/14). Half include psychology input (7/14). In addition, three services reported being able to refer to, or having the involvement of differing resources including post intensive treatment nursing teams, therapy assistant practitioners, outpatient services for people with neurological conditions, spiritual care teams, and specialist rehabilitation medical consultant services.\n\nMost long Covid rehabilitation services are delivered through a combination of face-to-face and digital contact (13/14). While precise numbers were not available, respondents reported large variations in the percentage of rehabilitation being delivered through the different forms of delivery, depending on clinical need. One respondent reported that their primary delivery route was digital. Another reported only delivering long Covid rehabilitation face-to-face, with no digital service.\n\nAlmost all respondents (13/14) reported patients being able to access long Covid rehabilitation through either hospital or GP referral. Many respondents stated that patients could also access long Covid rehabilitation through self-referral (11/14). Some respondents (3/14) stated other routes of access to long Covid rehabilitation including interdisciplinary referrals from other allied health professionals and social care, as well as referrals from informal carers. Responses on typical duration of rehabilitation were limited. Three respondents stated it was dependent on the individuals’ needs.\n\n\nDiscussion\n\nDespite some expert opinion that referral to community rehabilitation is not required for many people who have had COVID-1915, our findings demonstrate that community rehabilitation for people with long Covid is an emerging reality and is being provided across Scotland. Community rehabilitation for people with long Covid is currently being delivered predominantly by multidisciplinary teams of allied health professionals, with other specialists available as required. This is in keeping with community rehabilitation for other long-term conditions16. We have found variation in the modes in which long Covid rehabilitation is currently being delivered (face to face/digital/mixed) and provided (integrated services/new services) in Scotland. Symptoms that people with long Covid are presenting with to rehabilitation services are in keeping with the literature to date and provide an indication of the skill-mix and expertise required within a long Covid rehabilitation service. The wide range in numbers of patients accessing services is reflective of Scotland’s population density across its 14 territorial health boards, and of the range in infection rates across the country. Irrespective of the number of patients requiring rehabilitation for long Covid within a particular health board area, services will need to be able to provide appropriate and accessible rehabilitation, responsive to the diverse symptomatology and wider impact of the condition. Mode of delivery will be compounded by ongoing physical distancing measures.\n\nThis study has several limitations. We do not yet know which modes of delivery are most appropriate for this patient population. Innovative tele-health services are beginning to be proposed for this patient group17,18, and the one Health Board in our survey that reported developing a new long Covid specific service, described providing a predominantly digital service. Data on rehabilitation services in Scotland is not routinely collected, so detailed information on the numbers of referrals of people experiencing long Covid, the problems with which they were presenting, duration of rehabilitation, and specific interventions delivered was unavailable.\n\nThis survey provides a national picture of current community rehabilitation for people with long Covid symptomatology. To the best of our knowledge this is the first national survey of its kind. There is still lots to learn about current practice. While data on numbers of referrals, and content and duration of rehabilitation was requested, this information was not available to most of the respondents. A description of current services also does not provide any information on the effectiveness of the community rehabilitation service for people with diverse presentations, or of its perceived acceptability by its recipients. Detailed data on services and their recipients is vital and urgently required, to guide effective and efficient clinical practice and service planning and delivery.\n\nSeveral UK bodies and individuals have published expert opinion recommending a stepped, needs-based community rehabilitation approach incorporating information provision, self-management support and specialist services as required. They also recommend that rehabilitation should be individualised, progressive and utilise digital solutions19–21. How to optimise delivery of community rehabilitation is unknown, but vital to determine, given that rehabilitation services need to cope with additional COVID-19 demand whilst continuing to provide rehabilitation for other, often vulnerable, patient populations21.\n\nCommunity rehabilitation is a complex intervention22, which is provided in different ways according to clinical need, geographical location and financial costs. This complexity is further exacerbated when treating people with long Covid where the impact of the clinical sequalae is still unknown. While community rehabilitation has been routinely provided within the NHS in Scotland for many years, there are many unknowns regarding the delivery of community rehabilitation for people with long Covid. Therefore, research is urgently required to evaluate which models of community rehabilitation work, in what circumstances, and with whom.\n\n\nConclusions\n\nThis paper reports the findings of a national survey of current community rehabilitation provision for people with long Covid in Scotland. Almost all current services are providing a community rehabilitation response within current service provision. There is variation in the way in which these services are provided. Some information was unavailable due to the lack of routine data collection. With growing numbers of people presenting with symptoms of long Covid, further research is urgently required to investigate the implementation, outcomes and cost-effectiveness of differing models of community rehabilitation for this patient population.\n\n\nData availability\n\nDataSTORRE: Stirling Online Repository for Research Data. Survey Data for Long covid rehabilitation study. http://hdl.handle.net/11667/164\n\nThis project contains the following underlying data\n\n- Anonymised survey responses in .xlsx format\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nDataSTORRE: Stirling Online Repository for Research Data. Survey Data for Long covid rehabilitation study. http://hdl.handle.net/11667/165\n\nThis project contains the following underlying data\n\n- A copy of the survey sent to participants in .pdf format\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nThis paper originated from discussions between the authors. We would like to acknowledge and thank: Bette Locke (Scottish Government’s Professional Advisor for Rehabilitation), Dr Lesley Holdsworth OBE (National Clinical lead for Digital Health and Care), June Wylie (Professional Lead, Allied Health Professions, Healthcare Improvement Scotland), and Allan Cowie (Director of Services of Chest Heart and Stroke Scotland) for their helpful comments on the initial draft of the survey. We also thank all the survey respondents for their time and effort.\n\n\nReferences\n\nWorld Health Organisation: WHO Corona virus Disease (COVID-19 Dashboard. [Accessed on 09/1/20]. Reference Source\n\nBarker-Davies RM, O’Sullivan O, Senaratne KPP, et al.: The Stanford Hall consensus statement for post-COVID-19 rehabilitation. Br J Sports Med. 2020; 54(16): 949–959. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGreenhalgh T, Ladds E, Knight M, et al.: “Long Covid”: evidence, recommendations and priority research questions. Written evidence (COV0050) House of Lords Enquiry. 2020. Reference Source\n\nTenrforde MW, Kim SS, Lindsell CJ, et al.: Symptom Duration and Risk Factors for Delayed Return to Usual Health Among Outpatients with COVID -19 in a Multistate Health Care Systems Network – United States, March – June 2020. MMWR Morb Mortal Wkly Rep. 2020; 69(30): 993–998. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGreenhalgh T, Knight MA, Court C, et al.: Management of post-acute covid-19 in primary care. BMJ. 2020; 370: m3026. PubMed Abstract \| Publisher Full Text\n\nScottish Government: Scotland’s Digital Heath & Care Strategy. The Scottish Government, April 2018. Available from: [Accessed 2nd November 2020]. Reference Source\n\nLong Covid Support.[Accessed 2nd November 2020]. Reference Source\n\nPatient-Led Research Team: What Does COVID-19 Recovery Actually Look Like? An Analysis of the Prolonged COVID-19 Symptoms Survey. 2020. Reference Source\n\nMaxwell E: Living with Covid 19. A dynamic review of the evidence around ongoing Covid 19 symptoms (often called Long Covid). NIHR Centre for Engagement and Dissemination, September 2020. [Accessed 25th November 2020]. Reference Source\n\nSheehy LM: Considerations for Postacute Rehabilitation for Survivors of COVID-19. JMIR Public Health Surveill. 2020; 6(2): e19462. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAndrenelli E, Negrini F, de Sire A, et al.: Systematic rapid living review on rehabilitation needs due to COVID-19: update to May 31st, 2020. Eur J Phys Rehabil Med. 2020; 56(4): 508–514. PubMed Abstract \| Publisher Full Text\n\nGuttenbruner C, Stikes EK, Dreinhofer K: Why rehabilitation must have priority during and after the COVID-19 pandemic: A position statement of the global rehabilitation alliance. J Rehabil Med. 2020; 52(7): jrm00081. PubMed Abstract \| Publisher Full Text\n\nGrimshaw J: Surge (the survey reporting guideline). Guidelines for Reporting Health Research: A User's Manual. 2014 Jul 25; 206–13. Publisher Full Text\n\nHoffmann TC, Glasziou PP, Boutron I, et al.: Better reporting of interventions: template for intervention description and replication (TIDieR) checklist and guide. BMJ. 2014; 348: g1687. PubMed Abstract \| Publisher Full Text\n\nGreenhalgh T, Knight M, Buxton M, et al.: Management of post-acute covid-19 in primary care. BMJ. 2020; 370: m3026. PubMed Abstract \| Publisher Full Text\n\nBettger JA, Stineman MG: Effectiveness of multidisciplinary rehabilitation services in postacute care: state-of-the-science. A review. Arch Phys Med Rehabil. 2007; 88(11): 1526–34. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSalawu A, Green A, Crooks MG, et al.: A proposal for multidisciplinary tele-rehabilitation in the assessment and rehabilitation of COVID-19 survivors. Int J Environ Res Public Health. 2020; 17(13): 4890. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGonçalves AC, Leckie T, Hunter A, et al.: Technology supported rehabilitation for patients of critical illness caused by COVID-19: a protocol for a mixed-methods feasibility study. Int J Ther Rehabil. 2020; 27(10): 1–9. Publisher Full Text\n\nSheehy LM: Considerations for Postacute Rehabilitation for Survivors of COVID-19. JMIR Public Health Surveill. 2020; 6(2): e19462. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAndrenelli E, Negrini F, de Sire A, et al.: Systematic rapid living review on rehabilitation needs due to COVID-19: update to May 31st 2020. Eur J Phys Rehabil Med. 2020; 56(4): 508–514. PubMed Abstract \| Publisher Full Text\n\nGuttenbruner C, Stokes EK, Dreinhöfer K, et al.: Why rehabilitation must have priority during and after the COVID-19 pandemic: A position statement of the global rehabilitation alliance. J Rehabil Med. 2020; 52(7): jrm00081. PubMed Abstract \| Publisher Full Text\n\nRoberts JL, Din NU, Williams M, et al.: Development of an evidence-based complex intervention for community rehabilitation of patients with hip fracture using realist review, survey and focus groups. BMJ Open. 2017; 7(10): e014362. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "75829", "date": "09 Feb 2021", "name": "Suzanne McDonough", "expertise": [ "Reviewer Expertise Rehabilitation" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an important and timely piece of work which provides an overview, at a national level, of community based rehabilitation for people with long Covid, and key gaps in knowledge.\nStrengths of the work are the survey design i.e. using Surge guidelines for reporting and TIDIER as a structure for the questions with a combination of closed and open narrative questions; the team piloted the tool prior to use and involved key stakeholders in its development. It is a well written manuscript with use of key recent supporting references. There was an excellent overall response rate to the survey, and within the survey to the individual closed questions. The response to some open questions was good, and is absent for others. Overall this work is an important first step to inform service provision for managing people with long Covid.\nItems to consider addressing:\nWas there a plan to collate the responses to the narrative questions? and if so please add this to your data analysis section.\nPlease clarify in your methods:\nWere there reminders to respondents to complete the questionnaire or a single invitation only?\n\nDid the team consider other ways of enhancing the responses to the narrative questions? Might their open approach be a limitation to their survey? Do they think further closed questions with options may have helped, or a qualitative interview/focus group follow up?\n\nIn the discussion, first paragraph, you state that there was a wide variation in the numbers accessing services, this needs to be qualified as I understand from your results that this data was only available for 2/14 of the respondents so is largely unknown.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6465", "date": "26 Mar 2021", "name": "Edward Duncan", "role": "Author Response", "response": "Dear Professor McDonough, Thank you for taking the time to review this paper and for your helpful comments. Our responses to your review are below. Items to consider addressing: Was there a plan to collate the responses to the narrative questions? and if so please add this to your data analysis section. RESPONSE: Narrative responses were mostly short statements and insufficient to conduct for a formal thematic analysis or other qualitative method. Instead two members of the research team (ED, KC) reviewed responses and descriptively report them in the paper where they related to the study results. We have added this clarification to the methods section of our paper. Please clarify in your methods: Were there reminders to respondents to complete the questionnaire or a single invitation only? RESPONSE: As stated in the methods section, under \"data analysis\" - reminders were sent to non respondents after 1 week. Did the team consider other ways of enhancing the responses to the narrative questions? Might their open approach be a limitation to their survey? Do they think further closed questions with options may have helped, or a qualitative interview/focus group follow up? RESPONSE: Following up this survey with qualitative interviews or a focus group would have provided richer and more in-depth information, however these options were not feasible within the timeframe that was available. Further research into long Covid rehabilitation is now being conducted by the research team which includes interview and focus groups methods and we are confident that these methods will enhance our understanding of current community rehabilitation for people experience long covid. We have added this clarification to the methods section of our paper. In the discussion, first paragraph, you state that there was a wide variation in the numbers accessing services, this needs to be qualified as I understand from your results that this data was only available for 2/14 of the respondents so is largely unknown. RESPONSE: We have removed this sentence in response to your feedback." } ] }, { "id": "77654", "date": "16 Feb 2021", "name": "Lynette Mackenzie", "expertise": [ "Reviewer Expertise Community based occupational therapy with older people and people with chronic conditions." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI found this article an interesting and important read. I was wondering how a person with long COVID is identified - especially if their symptoms persist over 3 months after their COVID infection, and whether or not their access to rehabilitation would be dependent on them complaining of symptoms to someone who could refer such as their GP. This might mean that there are not the full numbers of people with long COVID who would be presenting to rehabilitation services which would limit the feedback that such services could provide in this survey.\nI was also interested that it was only the lead allied health people who were surveyed (n=14) since these people might not have a full idea of what services are actually provided at the coal face, being in more management positions. It would be interesting to send a similar survey to all community rehabilitation health professionals to get more detailed information. I'm sure the rural/urban differences would be interesting to look at with a larger sample.\n\nHaving said that, all these limitations were adequately addressed by the authors. I'm sure there will be further work done in this area.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6466", "date": "26 Mar 2021", "name": "Edward Duncan", "role": "Author Response", "response": "Dear Dr MacKenzie, Many thanks for your review and positive feedback. In response to another reviewer we have added a comment in the limitations of our revised paper, which highlights that some of our future research will investigate the issues that you raise." } ] } ]	1	https://f1000research.com/articles/9-1416
https://f1000research.com/articles/10-25/v1	15 Jan 21	{ "type": "Research Article", "title": "Use of chicken feather meal fermented with Bacillus subtilis in diets to increase the digestive enzymes activity and nutrient digestibility of silver pompano Trachinotus blochii (Lacepede, 1801)", "authors": [ "Adelina Adelina", "Feli Feliatra", "Yusni Ikhwan Siregar", "Iskandar Putra", "Indra Suharman", "Adelina Adelina", "Feli Feliatra", "Yusni Ikhwan Siregar", "Iskandar Putra" ], "abstract": "Background: Feather has the potential to be used as a fish feed ingredient because it has high protein content (80-85%), and is rich in amino acids arginine, leucine, isoleucine and valine. However, the protein consists mainly of keratin, which is classified as fiber that is difficult to digest. Therefore, to improve digestibility, the keratin protein is degraded using microbial Bacillus subtilis. This study aimed to determine the digestibility of fermented feather meal (FFM) in silver pompano diets and to observe the histological structure of their intestines after digestion. Methods: The method used was a one factor experiment with five treatments and three replications each, which were: diet without FFM (P0), diet containing 10% FFM (P1), 20% (P2), 30% (P3) and 40% (P4). The diets were given to juvenile silver pompano (with average body weight of 8.56 ± 0.18 g) and stocked with 10 fish in a 100 L capacity container. The experimental diets were given three times daily at approximately 8.00 AM, 12.00 PM and 5.00 PM to apparent satiation for 60 days. Results: The results showed that the use of FFM increased the activity of digestive enzymes (protease and lipase), but reduced the amylase activity of silver pompano, which was significantly different between treatments (P <0.05). Meanwhile, the diet containing 20% FFM produced the highest feed and protein, which are 37.05% and 67.24%, respectively. This was significantly different from other treatments (P <0.05), and was effectively absorbed by fish intestines. Conclusion: The addition of chicken feather meal fermented with Bacillus subtilis could increase the activity of digestive enzymes and nutrient digestibility of silver pompano.", "keywords": [ "Bacillus subtilis", "digestibility", "diet", "feather meal", "Trachinotus blochii" ], "content": "Introduction\n\nFeathers are by-products from chicken slaughterhouses, and production has continued to increase along with the rise in population and increased demand for chicken as a source of animal protein. Feather waste with poor management causes problems for human health because feathers are a source of odors and disease. Feathers are very difficult to degrade, and the decomposition process takes a long time, which has an impact on soil quality1.\n\nUtilization of feather waste is not optimal, and thus far, only a small portion is used to make dusters, vehicle seats, plant fertilizer, handicrafts, and shuttlecocks. In terms of nutrient content, feather meals are high in protein (80–85%) and rich in amino acids arginine, cystine, leucine, isoleucine, and valine2. Hence, they have the potential to be used as fish feed ingredients. However, 90% of the protein is composed of beta-keratin, a component that is classified as a difficult to digest fiber3. Keratin consists of cystine disulfide and hydrogen bonds, as well as hydrophobic interactions. The disulfide bonds between the cystine amino acids make the protein difficult to digest by proteolytic enzymes4. Therefore, to utilize feather as fish feed ingredient, the keratin needs to be degraded first.\n\nHeterotrophic bacteria found in the ocean have the potential to break down complex organic matter into simpler components5. In addition, a hydrolysis technique using microbes has been found to degrade keratin in feather protein using Bacillus licheniformis2 and Bacillus subtilis6. The keratinase enzyme produced by B. licheniformis can also hydrolyze various proteins including collagen, elastin, and keratin, as well as increase the digestibility of feather meal protein2. High or low digestibility of feather meal in feed is largely determined by the role of digestive enzymes. Bacteria of the genus Bacillus have a major role in the digestive tract of fish7 because they produce several extracellular enzymes such as proteases, lipases, and amylases. These enzymes catalyze the breakdown of complex nutrients (proteins, lipids and carbohydrates) into simple components, thereby increasing digestibility6. Adelina et al.8 showed that the digestibility of feed protein containing feather meal in white snapper (Lates calcarifer, Bloch) was 39.09%, increasing to 48.75% after fermentation using Bacillus subtilis.\n\nMeasuring the performance of feed absorption in fish digestive systems involves observing the histological structure of the intestine, which is an important organ responsible for digestion and absorption9. In addition, feed of good quality is not only based on the nutritional constituents, but also on other components that aid easy digestion and absorption. As long as the food is in the intestine, the nutrients are broken down by various enzymes into a form that can be absorbed, and then enter the circulatory system for use in physiological processes and growth.\n\nTherefore, this study aimed to examine the digestibility and absorption of feed containing fermented feather meal (FFM) in the intestine of silver pompano (Trachinotus blochii, Lacepede). This fish was chosen because of its important economic value; its market demand is quite high at local and international levels, particularly in Singapore, Japan, Taiwan, Hong Kong, and China10.\n\n\nMethods\n\nEthical approval for the study was obtained from Syiah Kuala University Research and Ethics Guidelines, Section of Animal Care and Use in Research (Ethic Code No: 958 /2015). All efforts were made to lessen harm to the animals by applying the Syiah Kuala University Research and Ethics Guidelines, Section of Animal Care and Use in Research ethical code.\n\nBroiler chicken feathers were collected from the local market, washed with clean water, sterilized by steaming at 100°C for 15 minutes, cooled at room temperature, and then dried in an oven at 60°C for six hours. Dried chicken feathers were grinded using a disk mill into feather meal. The material used for fermentation was Bacillus subtilis (GenBank accession no. JX188065.1), which was collected in the Marine Microbiology Laboratory of the Faculty of Fisheries and Marine Science, University of Riau, Indonesia11. Isolated B. subtilis was vortexed for inoculum, then transferred by dropping as much as 50 μ into a nutrient agar (NA) medium. The bacteria were flattened using a sprier, then incubated for 24 hours. The bacteria were then purified four times on the NA medium to obtain pure B. subtilis colonies. Pure B. subtilis was transferred to nutrient broth liquid media for propagation. B. subtilis was then used as a fermenter for chicken feather fermentation. The fermentation process was carried out by sterilizing the feathers in an oven at 100°C for 15 minutes, then cooling. The sterile outcome was placed in five Petri dishes (2 g each), adding 10 ml of pure B. subtilis, and incubating at 50°C, pH 8 for 72 hours12. The FFM was then ready to be used as fish feed ingredient.\n\nThe experimental diet used was artificial feed with a protein content of about 40–41%. The ingredients were purchased from the local market and weighed according to the formulations in Table 1, mixed until homogeneous, molded into pellets, and then dried in an oven at 60°C. The dry pellets were then analyzed in accordance AOAC methods13. Crude protein (Nx6.25) was measured using the micro-Kjeldahl nitrogen determination method. Crude lipid was determined by the ether-extraction method. Moisture was determined by oven-drying at 105°C until a constant weight was achieved. Ash content was measured after placing the samples in a muffle furnace at 550°C for 24 hours. The results of the proximate composition are shown in Table 1.\n\n1 FFM = fermented feather meal.\n\n2 Vitamin mix (mg/100 g diet): thiamin 5.0; riboflavin 5.0; Ca-pantothenate 10.0; niacin 2.0; pyridoxin 4.0; biotin 0.6; folic acid 1.5; cyanocobalamin 0.01; inositol 200; ρ-aminobenzoic acid 5.0; menadion 4.0; vit A palmitate 15.0; chole-calciferol 1.9; α-tocopherol 20.0; cholin chloride 900.0.\n\n3 Mineral mix (mg/100 g diet): KH2PO4 412; CaCO3 282; Ca (H2PO4) 618; FeCl3.4H2O 166; ZnSO4 9.99; MnSO4 6.3; CuSO4 2; CuSO4.7H2O) 0.05; KJ 0.15; Dextrin 450; Cellulose 553.51.\n\n4 NFE = nitrogen-free extract; calculated = 100-(%CP+%CL+%moisture+%ash+%CF).\n\nThis experiment used a completely randomized design (CRD). Five levels of FFM were tested, namely: P0 (0% FFM), P1 (10% FFM), P2 (20% FFM), P3 (30% FFM), and P4 (40% FFM). Every treatment was replicated three times.\n\nTen juvenile silver pompano were purchased from Batam Marine Aquaculture Center (BPBL) located in Setoko islands, Batam city with mean weight of 8.56±0.18 g were placed in 100 L plastic tanks. They were then fed three times daily at 8.00 AM, 12.00 PM, and 17.00 PM until apparent satiation. All leftover feeds were collected after one hour, and subsequently, fresh feces were collected by siphoning after 4–5 hours. Feces were also collected every morning before feeding time. All fecal samples were pooled until sufficient amounts had been obtained for chemical analysis. They were then completely dried in an oven at 60°C, ground using a laboratory grinder, and kept in a refrigerator at 16°C until further analysis. The protein, lipid, carbohydrate, and chromic oxide content of the feed and feces were analyzed using AOAC methods13 as mentioned above. After 60 days of the feeding test, two fish were taken from each plastic tank, anesthetized with tricaine methanesulfonate (MS-222) and their intestines were removed and frozen at -80°C until analysis for the activity of protease, lipase, and amylase enzymes. Intestinal retrieval was carried out 18 hours after consumption of the last feed14.\n\na. Protease, lipase, and amylase enzymes activity of fish\n\nTwo fish were taken from each test or six fish from each treatment, and their intestines were removed. The intestine was weighed, and a Tris buffer solution was added (20 mM Tris HCl, 1 mM EDTA, 10 mM CaCl2, pH 7.5) at a ratio of 10%. It was then put into an effendorf tube and centrifuged at 12,000 rpm for 10 mins, at a temperature of 4°C. The supernatant was taken and the activities of protease, lipase, and amylase enzymes were analyzed. Protease activity was determined using the method that was previously used by Bergmeyer et al.15. 0.01 M borate buffer (pH 8) was added into 2% casein substrates. The mixture was incubated at 37°C for 10 min, mixed with 0.1 M TCA and re-incubated at 37°C for 10 min. The mixture was centrifuged at 4000 rpm for 10 min, mixed with 5.0 mL Na2CO3 and 1 mL reagent Follin (1:2). The absorbance was checked using spectrophotometer (UV-1800, Shimadzu Europa, Duisburg, Germany) at 578 nm. Lipase activity was analyzed according to Borlongan16. 1.5 mL olive oil was mixed with 1.5 mL of 0.1 M HCl tri buffer at pH 8.0. The mixture was incubated for 6 h at 37°C, pursued by adding 3 mL ethyl alcohol (95%). 0.01 N NaOH was applied to titrate the mixture by using 0.9% thymolphthalein ethanol as an indicator. Analysis of amylase activity was based on Bernfield17. The reaction mixture consisted of citra buffer solution (pH 5.7) and starch solution 1%. The mixture was incubated for 30 min at 20°C, mixed with 2 mL DNS and poached for 5 min. The absorbance was measured by using a spectrophotometer (UV-1800, Shimadzu Europa, Duisburg, Germany) at 550 nm.\n\nb. Fish intestine histology\n\nHistological analysis was conducted to observe the structure of the intestinal wall after being given a test feed containing FFM. At the end of maintenance, the intestines of the test fishes from each treatment were removed, and histologically prepared to observe the condition using the Olympus binocular microscope model CX 21. The observations were then compared to determine the differences between the treatments. The preparation method was carried out according to Dellman et al.18 The fish was dissected and their intestines were taken cut to a thickness of 0.5 cm and then fixed in 10% formalin solution for 24 hours. The dehydration process was carried out, which begins by putting the sample in a bottle containing 30%, 50%, 70%, 90% and 100% alcohol for 45 minutes each, which aims to remove the moisture content from the cells / tissues and replace it with alcohol. Then the sample was inserted into xylol 1 and xylol 2 for 45 minutes each for dealcoholization.\n\nEnzyme activity data (protease, lipase, and amylase) and feed digestibility were analyzed using analysis of variance (ANOVA). Duncan’s Multiple Range Test (DMRT) was used to determine the differences between the treatments. The analysis was performed using SPSS v. 18.0 software. Alphabetical notations (a, b, c) are used to mark significant differences at a significance level of p<0.05. Histology of the intestine was descriptively analyzed by comparing the conditions for each treatment.\n\n\nResults\n\nProtease, lipase, and amylase enzymes activity measurements in the intestine of silver pompano with FFM feed can be seen in Figure 1, Figure 2, and Figure 3, respectively. The results showed that protease and lipase enzyme activities were higher at the end of the observation compared to the beginning. In contrast, amylase activity was higher at the beginning than the end.\n\nP0 = FFM 0%, P1 = FFM 10%, P2 = FFM 20%, P3 = FFM 30%, and P4 = FFM 40%.\n\nP0 = FFM 0%, P1 = FFM 10%, P2 = FFM 20%, P3 = FFM 30%, and P4 = FFM 40%.\n\nP0 = FFM 0%, P1 = FFM 10%, P2 = FFM 20%, P3 = FFM 30%, and P4 = FFM 40%.\n\nProtease, lipase, and amylase enzyme activities in the intestine of fish fed with FFM (P1, P2, P3 and P4) were found to be higher than in those without FFM (P0) and were significantly different (p<0.05) between the treatments. The highest protease activity was found in the P3 (30% FFM) diet, which was significantly different from other treatments (p<0.05). The highest lipase and amylase enzyme activities were found in the P4 (40% FFM diet), which were significantly different from other treatments (p<0.05).\n\nFeed digestibility is strongly influenced by digestive enzymes that breakdown nutrients and make them easily absorbed. The feed containing FFM resulted in the levels of digestibility of proteins, lipids and carbohydrates shown in Table 2.\n\n*Data values are the mean and standard deviation. The means with different superscripts (a, b, c) in the same row were significantly different (p < 0.05).\n\nThe fermentation of feather meal using B. subtilis resulted in increased digestibility. The feed without FFM (P0) had the lowest digestibility compared to those that contain FFM (P1, P2, P3, and P4) and was significantly different from the other treatments (p<0.05). Feed with 20% FFM (P2) had the highest digestibility but was not significantly different (p>0.05) from feed containing 30% FFM (P3).\n\nThe use of different FFM percentages in feed resulted in different levels of digestibility of nutrients (proteins, lipids, and carbohydrates). The feed without FFM (P0) had the lowest protein digestibility compared to feed containing FFM (P1, P2, P3, and P4) and was significantly different from the other treatments (p<0.05). Meanwhile, the feed containing 20% FFM (P2) had the highest digestibility, and was significantly different (p<0.05) from the other treatments. 30% FFM (P3) and 40% FFM (P4) feeds had lower protein digestibility compared to those containing 20% FFM (P2). Feed without FFM (P0) had lipid digestibility that was not significantly different (p>0.05) from those with FFM (P1, P2, P3 and P4). In addition, feed without FFM (P0) had the lowest carbohydrate digestibility compared to feed with FFM (P1, P2, P3, and P4) and was significantly different from the other treatments (p<0.05), whereas feed containing FFM (P1, P2, P3 and P4) had carbohydrate digestibility that was not significantly different between treatments (p>0.05).\n\nThe intestinal histology of silver pompano fed with FFM is presented in Figure 4.\n\nTMK = tunica muscularis, SP = leftover feed, SG = goblet cells (mucous cells), K = congestion, H = hemorrhage, I = irritation/erosion, N = necrosis coloration, HE = Hematoxylin Eosin, enlargement 1000x.\n\nFigure 4 shows that fish fed without FFM (P0) have normal intestinal conditions and can easily digest food. The intestine of P1 treated fish (10% FFM) was seen to produce goblet cells that facilitate absorption. Those fed with 20% FFM (P2) produced more goblet cells in the intestine, suggesting they are able to effectively absorb feed. The use of 30% FFM in feed (P3) resulted in congestion (capillary tissue full of blood) and hemorrhage (blood spreads to the tissues) in the intestinal tissue of the fish. This is characterized by erythrocytes that leak out of blood vessels into the mucus membrane of intestinal tissue. Furthermore, the use of more FFM in feed (40%, P4) caused damage to the intestinal tissue (necrosis), and the villous wall became irritated. The rest of the feed was seen accumulating in the intestine, which suggests that feed containing high FFM (40%) is not properly absorbed, but rather causes intestinal damage.\n\n\nDiscussion\n\nProtease and lipase enzyme activity was higher at the end of the experiment than at the beginning. This is in contrast to amylase, for which activity was higher at the beginning19. The activity of protease, lipase, and amylase enzymes of silver pompano can be detected during the larva stage, and tends to increase with age20. Protease activity in batik grouper larvae (Epinephelus microdon) was shown to increase with age21. Enzyme activity tends to increase with fish age because with age the digestive organs develop and enter the definitive phase; therefore, the consumption of exogenous feed is increased, which is a source of energy to trigger enzyme activity. In addition, high activity is related to the role of the pancreas in secreting enzymes. When an enzyme is secreted in small amounts, the activity will be low. However, when it is secreted in large amounts, the activity will increase.\n\nAmylase activity was seen to be lower at the end than at the beginning of the observation19. The activity increased up to the 24th day, then decreased to the 30th day of observation14. The decrease in enzyme activity is influenced by the length of its reaction with the substrate. A longer reaction causes the enzyme to lose some of its activity. The reduced amylase activity in fish is also influenced by low concentrations in the digestive tract or concentrations that have exceeded the optimal limit. Hence, even though the fish consumes a high amount of the substrate, as long as the amylase concentration is low, the activity will not increase.\n\nThe results also showed that the protease enzyme activity of fish fed with FFM (P1, P2, P3 and P4) was higher than those without FFM (P0), and was significantly different (P <0.05) between the treatments. The highest protease enzyme activity was found in 30% FFM feed and was significantly different from other treatments (p<0.05). Jayadi20 stated that the increase in protease activity is caused by the exogenous feed consumed, which stimulates the increase in digestive enzyme activity. The production of these enzymes is strongly influenced by the amount of protein in the feed. In addition, Lundstedt et al.22 stated that protease activity is strongly influenced by the amount of active protease, the feed, and the quality of the diet.\n\nThe feed that contained no FFM in this study was able to stimulate an increase in lipase enzyme activity. However, the more FFM (40%) in the feed, the higher the lipase activity was found to be, and activity was significantly different between treatments (p<0.05). This is supported by Duc et al.23, who stated that some Bacillus species produce extracellular enzymes such as proteases, lipases, amylase, and cellulases that facilitate feed digestion. In addition, several factors that influence enzyme activity according to Yamin and Palinggi14 are the nature of the substrate, the type of enzyme, and the environmental conditions in the digestive tract.\n\nAmylase activity was higher with increasing FFM in the feed, and was significantly different (p<0.05) between treatments. Chor et al.24 stated that activity is influenced by feed composition, carbohydrate content, and fish feeding habits. Chor et al.24 also stated that the lipase and protease activity of omnivorous fish including star pomfret was higher than amylase. Furthermore, omnivorous species of fish have amylase levels and an amylase-protease ratio that is higher than those of carnivorous types.\n\nFeed without FFM (P0) had the lowest digestibility compared to those with FFM (P1, P2, P3, and P4). Feed containing 20% FFM (P2) had the highest digestibility but was not significantly different (p>0.05) from feed containing 30% FFM (P3). Mazotto et al.4 and Brandelli et al.6 stated that B. subtilis produces protease, lipase, and amylase that are useful in improving the quality of proteins and breaking down complex nutrients into simple absorbable molecules, thereby increasing digestibility in feed without FFM25. Digestibility values of artificial feed depends on the level of fish reception and the available enzymes. Therefore, the addition of exogenous enzyme has the potential to optimize feed digestibility. In addition, when the protein, lipid, and carbohydrate content correspond to the enzyme activity, digestibility will increase.\n\nFeed that contained FFM had a higher protein digestibility than those without it. This is supported by Zerdani et al.26, who stated that processing feather meal with B. licheniformis increased the digestibility of the protein by 54.20%. The results of this study showed that feed and protein digestibility are best obtained in fish fed with 20% FFM (P2)27. The digestibility of proteins is determined by exogenous and endogenous factors. The exogenous factors are the interactions of proteins with polyphenols, carbohydrates, lipids, and protease inhibitors. The endogenous factors are related to the characterization of protein structures such as tertiary, quaternary, and structures that can be damaged by heat. When compared with the results of Adelina et al.8, in which feed containing 10% FFM fermented by B. subtilis in white snapper produced 48.75% protein digestibility, the value of this study was higher (67.24%).\n\nFeed without FFM (P0) had lipid digestibility that was not significantly different (p>0.05) from that of feed containing FFM (P1, P2, P3 and P4). Marzuqi and Anjusary28 stated that high or low digestibility is influenced by lipase enzyme activity. Therefore, the more the lipid in the feed, the higher the activity of lipase. Furthermore, digestibility is influenced by several factors including protein and carbohydrate components, the process of making the feed, the particle size, type and size of fish and the amount of feed consumed25.\n\nFeed without FFM (P0) had the lowest carbohydrate digestibility compared to those containing FFM (P1, P2, P3, and P4) and was significantly different (p<0.05), whereas those containing FFM (P1, P2, P3 and P4) had carbohydrate digestibility that was not significantly different between treatments (p>0.05). This correlated with higher amylase activity in fish fed with FFM compared to those without FFM. Marzuqi and Anjusary28 stated that fish do not have adequate carbohydrate digestive enzymes in their digestive tract, hence the digestibility value of this nutrient is generally low. However, FFM fermentation using B. subtilis in this study was able to produce higher amylase activity and increase the digestibility of carbohydrates.\n\nFish fed with no FFM (P0) showed a normal intestine on histological examination, and can therefore properly digest feed29. The intestine has an important role in food digestion, especially in nutrient absorption. Fujaya30 stated that nutrients are not directly absorbed, but are first broken down into their simpler components in the form of amino acids, fatty acids, and glucose. Specifically for proteins, the degradation process occurs in the stomach by pepsin and in the intestine by trypsin30. Meanwhile, fish intestine after treatment P1 (10% FFM) was seen to produce goblet cells. These cells produce mucus, which has a role in facilitating absorption and transportation of feed molecules through membranes, as well as providing protection against micro-organisms in the intestine31. The test feed containing 10% FFM was seen to be absorbed by the intestine of silver pompano9. Feather protein degraded by the B. subtilis enzyme could be digested and effectively absorbed by the intestines of broiler chickens, and did not damage their intestinal function. However, the intestine of P1 fish showed congestion. Kalaiyarasi et al.32 stated that this is an event of vessel dilation due to increased blood volume in the circulatory system.\n\nThe intestine of fish fed with a diet containing 20% FFM (P2) produced more goblet cells, which means they effectively absorb nutrients18. Fish digestive activity requires a lot of enzyme secretion; therefore, the intestine stimulates goblet cells to produce more mucus to protect the outer lining against damage and irritation33. Probiotic bacteria regulate the microbial environment in fish intestines and block pathogenic micro-organisms by releasing amylase, protease, cellulase, and lipase, which help to hydrolyze nutrients. They also facilitate the breakdown of carbohydrates, proteins, and lipid into smaller molecules.\n\nThe use of 30% FFM in feed (P3) resulted in congestion and hemorrhage of the fish intestinal tissue. This is characterized by erythrocyte leakage in the mucus membrane. The hemorrhage that occurs in the intestine can be caused by several agents such as foreign materials or objects that enter into the digestive tract, causing damage to the wall34.\n\nIn fish fed with 40% FFM (P4), the intestine had more severe damage (necrosis) and the villi wall was irritated. Furthermore, the rest of the feed piled up in the intestine, which showed that 40% FFM is quite high and is not properly absorbed, causing damage. The necrosis that occurs is characterized by the appearance of damaged tissue18. Necrosis is death of a cell or tissue after an advanced stage of degeneration. This can be caused by trauma or an interruption in blood supply in certain areas34. The necrotic tissues have several characteristics including an abnormal pale color, brittleness, and poor consistency. Hence, erosion or irritation of intestinal villi causes the loss of some epithelium in the lining of mucosa, causing it to become thinner. This will in turn cause disruption of nutrients absorption, leading to malnutrition and even death34.\n\n\nConclusion\n\nThe use of FFM in feed resulted in increased activity of digestive enzymes (protease, lipase, and amylase) in silver pompano. The more FFM there was in the feed, the higher the activities of the enzymes were. The digestibility of nutrients (proteins, lipids, and carbohydrates) in feed that contained FFM was better than in feed without FFM. While 20% FFM in feed was efficiently digested and absorbed, 40% FFM could not be digested or absorbed, but rather caused intestinal irritation and necrosis.\n\n\nData availability\n\nFigshare: Raw data of Cr2O3 and proximate composition of diets. https://doi.org/10.6084/m9.figshare.13109486.v135.\n\nFigshare: Raw data of enzyme activity. https://doi.org/10.6084/m9.figshare.13109495.v136.\n\nFigshare: Raw data of feed and nutrient digestibility. https://doi.org/10.6084/m9.figshare.13109501.v137.\n\nFigshare: Intestinal histology. https://doi.org/10.6084/m9.figshare.13109498.v138.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nOkareh OT, Awe AO, Sridhar MKC: Effect of processed feather waste as mulch on crop growth and soil fertilization. J Agric and Eco Res Int. 2015; 4(1): 25–35. Publisher Full Text\n\nTiwary E, Gupta R: Rapid conversion of chicken feather to feather meal using dimeric keratinase from Bacillus licheniformis ER-15. J Bioproces Biotechniq. 2012; 2: 4. Publisher Full Text\n\nPandian C, Pandiyan MT, Sundaresan A, et al.: Haematological profile and erythrocyte indices in different breeds of poultry. Int J Livest Res. 2012; 2(3): 89–92. Reference Source\n\nMazotto AM, Coelho RR, Cedrola SM, et al.: Keratinase production by three Bacillus spp. using feather meal and whole feather as substrate in a submerged fermentation. Enzyme Res. 2011; 2011: 523780. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFeliatra F, Effendi I, Yanti SL: Characteristic genetics of heterotrophic bacteria in Siak River Estuary, Riau Province, Indonesia as Prospective Anti-pathogenic Bacteria to Fish and Shrimps. J Pure Appl Microbiol. 2018; 12(4): 1801–1808. Publisher Full Text\n\nBrandelli A, Sala L, Kalil SJ: Microbial enzymes for bioconversion of poultry waste into added-value products. Food Res Int. 2015; 73: 3–12. Publisher Full Text\n\nFeliatra F, Hamdani R, Lukistyowati I, et al.: Sensitivity of Heterotrophic Bacteria in the Low-Salinity Water Areas and Estuary in Siak District toward Pathogenic Bacteria in Fish. Int J Microbiol. 2019; 2019: 7456410. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAdelina A, Feliatra F, Siregar Y, et al.: Utilization of feather meal fermented Bacillus subtilis to replace fish meal in the diet of silver pompano, Trachinotus blochii (Lacepede, 1801). AACL Bioflux. 2020; 13(1): 100–108. Reference Source\n\nAdejumo IO, Adetunji CO: Production and evaluation of biodegraded feather meal using immobilised and crude enzyme from Bacillus subtilis on broiler chickens. Brazillian Journal of Biological Sciences. 2018; 5(10): 405–416. Publisher Full Text\n\nRansangan J, Abdullah A, Roli Z, et al.: Betanoda virus infection in Golden pompano, Trachinotus blochii fingerlings cultured in deep-sea cage culture facility in Langkawi, Malaysia. Aquaculture. 2011; 315(3/4): 327–334. Publisher Full Text\n\nFeliatra F, Lukistyowati I, Yoswaty, et al.: Phylogenetic analysis to compare populations of acid tolerant bacteria isolated from the gastrointestinal tract of two different prawn species Macrobrachium rosenbergii and Penaeus monodon. AACL Bioflux. 2016; 9(2): 360–367. Reference Source\n\nDesi M: Bacillus licheniformis keratinase activity in breaking keratin chicken feathers. BSc Thesis, Faculty of Math and Science, Bogor Agricultural University, Bogor. [in Indonesia.]. 2002; 13.\n\nAOAC: Official methods of analysis of AOAC International. 19th edition, AOAC 53 International, Gaithersburg, Maryland, USA. 2012; 1263. Reference Source\n\nYamin M, Palinggi NN: Protease enzyme activity and digestive conditions in the gut of tiger grouper (Epinephelus fuscoguttatus) after feeding. Jurnal Riset Akuakultur. [in Indonesian]. 2007; 2(2): 281–288. Reference Source\n\nBergmeyer HU, Grassi M: Methods of enzymatic analysis. Vol. II. 3rd eds. Verlag Chemie, Weinhein. 1983; 539.\n\nBorlongan IG: Studies on the digestive lipases of milkfish, Chanos chanos. Aquaculture. 1990; 89(3-4): 315–325.\n\nBernfeld P: Amylase a and b. In: Methods in Enzymology, Colowick, S.P. and N.O. Kaplan (Eds.). Academic Press, New York. 1955; 149–152.\n\nDellman HD, Brown EM: Textbook of Veterinary histology. Eds. III. UI Press, Jakarta. 1992; 279. Reference Source\n\nPranata A, Haryati, Karim M Y: Development of enzyme activity in silver pompano larvae (Trachinotus blochii, Lacepede 1801). J Sci Technol. [in Indonesian]. 2014; 14(3): 199–208. Reference Source\n\nJayadi: Biological and physiological aspects as well as environmental requirements and larva of the batik grouper (Epinephelus microdon)]. Postgraduate Program. Hasanuddin University. Makassar. [in Indonesian]. 2004; 105. Reference Source\n\nMoguel-Hernandez I, Pena R, Nolasco-Soria H, et al.: Development of digestive enzyme activity in spotted rose snapper, Lutjanus guttatus (Steindachner, 1869) larvae. Fish Physiol Biochem. 2014; 40(3): 839–848. PubMed Abstract \| Publisher Full Text\n\nLundstedt LM, Melo JFB, Morales G: Digestive enzyme and metabolic profile Pseudoplatystoma corruscans (Teleostei: Siluriformes) in response to diet compositian. Comp Biochem Physiol B Biochem Mol Biol. 2004; 137(3): 331–339. PubMed Abstract \| Publisher Full Text\n\nDuc LH, Hong H A, Barbosa TM, et al.: Characterization of Bacillus probiotics available for human use. J Appl Environ Microbiol. 2004; 70(4): 2161–2171. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChor, WK, Lim LS, Shapawi R: Evaluation of feather meal as a dietary protein source for African catfish fry Clarias gariepinus. J Fish Aquat Sci. 2013; 8(6): 697–705. Publisher Full Text\n\nAlmeide DLC, Lundstedt LM, Moraes G: Digestive enzyme responses of tambaqui (Colossoma macropomum) fed on different levels of protein and lipid. Aquaculture Nutrition. 2006; 12(6): 443–450. Publisher Full Text\n\nZerdani I, Faid M, Malki A: Feather wastes digestion by new isolated strains Bacillus sp. in Morocco. African Journal of Biotechnology. 2004; 3(1): 67–70. Publisher Full Text\n\nGuo X, Yao H, Chen Z: Effect of heat, rutin and disulfide bond reduction on in vitro pepsin digestibility of Chinese tartary buckwheat protein factions. Food Chemistry. 2007; 102(1): 118–122. Publisher Full Text\n\nMarzuqi M, Anjusary DN: Digestion of feed nutrients with different levels of protein and lipid in juvenile grouper sand (Epinephelus corallicola). Jurnal Ilmu dan Teknologi Kelautan Tropis. [in Indonesian]. 2013; 5(2): 311–323.\n\nHaloi K, Kalita M, Nath R: The study on the histopathological changes of stomach of Channa punctatus Bloch. By used pesticide endosulfan. The Global Journal of Science Frontier Research Biological Sciences. 2013; 13(2): 1–6. Reference Source\n\nFujaya Y: Fish Fisiology: The basis of fisheries technology development. First print. Rineka Putra. Jakarta. [in Indonesian]. 2004; 165. Reference Source\n\nArman S, Ucuncu SI: Histochemical characterization of convict cichlid (Amatitlania nigrofasciata) intestinal goblet cells. Pakistan J Zool. 2017; 49(2): 417–424. Reference Source\n\nKalaiyarasi T, Jayakumar N, Jawahar P, et al.: Histological changes in the gill and liver of marine spotted Catfish, Arius maculatus from sewage disposal site, Therespuram off Thoothukudi Southeast Coast of India. J Entomol Zool Stud. 2017; 5(5): 1710–1715. Reference Source\n\nFarouq A: Application of probiotics, prebiotics and symbiotics in feed to improve the immune response and survival of Oreochromis niloticus tilapia infected by Streptococcus agalactiae. Bogor Agricultural University. Bogor. [in Indonesian]. 2011; 78.\n\nPlumb JA, Hanson LA: Health maintenance and principal microbial diseases of cultured fishes. CRC Press Inc. USA. 1994; 264. Publisher Full Text\n\nSuharman I: Raw data of Cr2O3 and proximate composition of diets. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.13109486.v1\n\nSuharman I: Raw data of enzyme activity. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.13109495.v1\n\nSuharman I: Raw data of feed and nutrient digestibility. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.13109501.v1\n\nSuharman I: Intestinal histology. figshare. Figure. 2020. http://www.doi.org/10.6084/m9.figshare.13109498.v1" }	[ { "id": "77534", "date": "08 Feb 2021", "name": "Christopher Marlowe Caipang", "expertise": [ "Reviewer Expertise Fish health management", "immunology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript describes the use of chicken feather meal fermented with Bacillus subtilis to increase digestibility and digestive enzyme activity in pompano. Results showed that FFM enhanced digestive protease and lipase but not amylase. Supplementation with 20% fermented FFM resulted in higher digestibility of nutrients.\nThe results presented are straightforward. However, there are certain issues in the methodology that I think the authors need to address:\nPlease provide data that the fermentation of feather meal with Bacilus resulted in the partial breakdown down the composition.\n\nFor the nutrient digestibility studies, how did the authors prevent leaching of the nutrients from feces during collection?\n\nThe initial levels of amylase were high. Theoretically, the levels of the digestive enzymes will be more or less similar with the P0 levels. In the case of amylase, there is a wide discrepancy between P0 and the initial. Please explain this phenomenon.\n\nKindly explain why no analyses were done within the 60 day of feeding. An analysis for 2-3 sampling points within the feeding period will give us a better picture of the trends of the enzyme activity.\n\nPlease explain why very few fish were used for the experiment.\n\nPlease provide a better resolution of the histological slides that will clearly show changes in some of the morphological/cellular structures to indicate the effect of the FFM.\nI hope that these issues will be addressed by the authors when they revise their manuscript.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6471", "date": "25 Mar 2021", "name": "Indra Suharman", "role": "Author Response", "response": "1. Comment: Please provide data that the fermentation of feather meal with Bacilus resulted in the partial breakdown down the composition. Respond: We have published the data of fermentation of feather meal in IOP Conference Series : Earth and Environmental Science (2019). 2. Comment: For the nutrient digestibility studies, how did the authors prevent leaching of the nutrients from feces during collection? Respond: To prevent leaching of the nutrients from feces during collection, we directly collected the feces that are released by the fish by siphoning (siphoning method). 3. Comment: The initial levels of amylase were high. Theoretically, the levels of the digestive enzymes will be more or less similar with the P0 levels. In the case of amylase, there is a wide discrepancy between P0 and the initial. Please explain this phenomenon. Respond: This phenomenon might be affected by feeding habits and biochemical composition of diet. Other studies indicated that the amylase activity largely depends on the feeding habits of fish. We have added more discussion in the discussion section. 4. Comment: Kindly explain why no analyses were done within the 60 day of feeding. An analysis for 2-3 sampling points within the feeding period will give us a better picture of the trends of the enzyme activity. Respond: Because we only want to know the enzyme activity at the beginning and the end of the experiment. 5. Comment: Please explain why very few fish were used for the experiment. Respond: We have used a total of 150 fish that were distributed into 15 identical 20-L plastic jars at a density of 10 fish per jar. We have made a corrrection in the material and method section. 6. Comment: Please provide a better resolution of the histological slides that will clearly show changes in some of the morphological/cellular structures to indicate the effect of the FFM. Respond: We only have this resolution of the histological slides." } ] }, { "id": "79933", "date": "05 Mar 2021", "name": "Rudy Agung Nugroho", "expertise": [ "Reviewer Expertise Animal/Fish Physiology", "Fish nutrition", "Biochemistry." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nReview report for the work entitle: Use of chicken feather meal fermented with Bacillus subtilis in diets to increase the digestive enzymes activity and nutrient digestibility of silver pompano Trachinotus blochii (Lacepede, 1801). This work is informative, has a great result, and benefit for the knowledge of fisheries fields. However, there are some points should be addressed to the author(s) and need to be clarified.\nTitle\nAbstract Background: please add scientific name of silver pompano for the first appear in this section.\nMethods: the term P1, P2, P3, P4 can be deleted, it is unused in this abstract.\nConclusion, please be more specific, it is stated that the addition of Chicken feather meal fermented with B. subtilis could increase the activity of digestive enzyme, but the result found that the amylase activity of silver pompano was reduced.\nIntroduction Acceptable.\nMaterials and Methods\nIt is stated that “ The sterile outcome was placed in five Petri dishes (2 g each), adding 10 ml of pure B. subtilis, and incubating at 50°C, pH 8 for 72 hours 12. The FFM was then ready to be used as fish feed ingredient.” Does the FFM that was resulted from five Petri dishes was enough for the whole study?\n\nPlease mention the density of fish in each plastic tank, and how much water in each plastic tank.\n\nPlease be specific on how the protease, amylase and lipase activity were determined, there should be any of equations.\n\nIn analysis of intestine histology: it is mention that the observations were then compared to determine the differences between the treatments. Please state what variable that was compared among the treatments (Goblet, histopathology?).\nResults Adequate.\nDiscussion Please discuss why amylase activity was lower at the end of the study in relation with FFM. This in contrast with the statement that feed with FFM had the higher digestibility, though was not significantly different among treatments.\nConclusion Sufficient.\nReferences Sufficient.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6464", "date": "25 Mar 2021", "name": "Indra Suharman", "role": "Author Response", "response": "1. Comment : Please add scientific name of silver pompano for the first appear in this section. Respond : We have added this information in the background of abstract. 2. Comment : The term P1, P2, P3, P4 can be deleted, it is unused in this abstract. Respond : We have deleted this information in the method of abstract. 3. Comment : Please be more specific, it is stated that the addition of Chicken feather meal fermented with B. subtilis could increase the activity of digestive enzyme, but the result found that the amylase activity of silver pompano was reduced. Respond : We have explained more specific this statement in the conclusion of abstract. 4. Comment : It is stated that “ The sterile outcome was placed in five Petri dishes (2 g each), adding 10 ml of pure B. subtilis, and incubating at 50°C, pH 8 for 72 hours 12. The FFM was then ready to be used as fish feed ingredient.” Does the FFM that was resulted from five Petri dishes was enough for the whole study? Respond : We only used the FFM that was resulted from five petri dishes for the proximate composition analysis after fermentation. 5. Comment : Please mention the density of fish in each plastic tank, and how much water in each plastic tank. Respond : We have added this information in the material and method section. 6. Comment : Please be specific on how the protease, amylase and lipase activity were determined, there should be any of equations. Respond : We have added this information in the material and method section. The activity of protease, amylase and lipase enzymes were determined by following methods that were previously used by Bergmeyer et al. (1983), Borlongan (1990) and Bernfield (1955) respectively which the equations were involved in these methods. 7. Comment : In analysis of intestine histology: it is mention that the observations were then compared to determine the differences between the treatments. Please state what variable that was compared among the treatments (Goblet, histopathology?). Respond : We have mentioned this information in the material and method section. 8. Comment : Please discuss why amylase activity was lower at the end of the study in relation with FFM. This in contrast with the statement that feed with FFM had the higher digestibility, though was not significantly different among treatments. Respond : We have developed the discussionas suggested by the reviewer in the discussion section." } ] } ]	1	https://f1000research.com/articles/10-25
https://f1000research.com/articles/9-1395/v1	03 Dec 20	{ "type": "Research Article", "title": "In silico prediction of structure and function for a large family of transmembrane proteins that includes human Tmem41b", "authors": [ "Shahram Mesdaghi", "David L. Murphy", "Filomeno Sánchez Rodríguez", "J. Javier Burgos-Mármol", "Daniel J. Rigden", "Shahram Mesdaghi", "David L. Murphy", "Filomeno Sánchez Rodríguez", "J. Javier Burgos-Mármol" ], "abstract": "Background: Recent strides in computational structural biology have opened up an opportunity to understand previously uncharacterised proteins. The under-representation of transmembrane proteins in the Protein Data Bank highlights the need to apply new and advanced bioinformatics methods to shed light on their structure and function. This study focuses on a family of transmembrane proteins containing the Pfam domain PF09335 ('SNARE_ASSOC'/ ‘VTT ‘/’Tvp38’). One prominent member, Tmem41b, has been shown to be involved in early stages of autophagosome formation and is vital in mouse embryonic development as well as being identified as a viral host factor of SARS-CoV-2. Methods: We used evolutionary covariance-derived information to construct and validate ab initio models, make domain boundary predictions and infer local structural features. Results: The results from the structural bioinformatics analysis of Tmem41b and its homologues showed that they contain a tandem repeat that is clearly visible in evolutionary covariance data but much less so by sequence analysis. Furthermore, cross-referencing of other prediction data with covariance analysis showed that the internal repeat features two-fold rotational symmetry. Ab initio modelling of Tmem41b and homologues reinforces these structural predictions. Local structural features predicted to be present in Tmem41b were also present in Cl-/H+ antiporters. Conclusions: The results of this study strongly point to Tmem41b and its homologues being transporters for an as-yet uncharacterised substrate and possibly using H+ antiporter activity as its mechanism for transport.", "keywords": [ "ab initio modelling", "bioinformatics", "autophagy", "contact predictions", "evolutionary covariance", "DedA", "SARS-CoV-2", "Tmem41b", "VTT domain" ], "content": "Introduction\n\nA protein’s structural information is crucial to understand it’s function and evolution. Currently, there is only experimental structural data for a tiny fraction of proteins (Khafizov et al., 2014). For instance, membrane proteins are encoded by 30% of the protein-coding genes of the human genome (Almén et al., 2009), but they only have a 2% representation in the Protein Data Bank (PDB) (Koehler Leman et al., 2015). Membrane protein families are particularly poorly understood due to experimental difficulties, such as over-expression, which can result in toxicity to host cells (Grisshammer & Tateu, 1995), as well as difficulty in finding a suitable membrane mimetic to reconstitute the protein. Additionally, membrane proteins are much less conserved across species compared to water-soluble proteins (Sojo et al., 2016), making sequence-based homologue identification a challenge, and in turn rendering homology modelling of these proteins more difficult. Membrane proteins can be grouped according to their interaction with various cell membranes: integral membrane proteins (IMPs) are permanently anchored whereas peripheral membrane proteins transiently adhere to cell membranes. IMPs that span the membrane are known as transmembrane proteins (TMEMs) as opposed to IMPs that adhere to one side of the membrane (Fowler & Coveney, 2006).\n\nOne IMP protein family is Tmem41, which has two human representatives, namely Tmem41a and Tmem41b; both share the PF09335 ('SNARE_ASSOC'/ ‘VTT ‘/’Tvp38’) Pfam (El-Gebali et al., 2019) domain. The profile of Tmem41b has recently risen due to experimental evidence pointing to its involvement in macroautophagy regulation (making it a possible Atg protein, i.e. an autophagy related protein) and lipid mobilisation (Moretti et al., 2018). Other studies identify Tmem41b to be involved in motor circuit function, with TMEM41B-knockout Drosophila showing neuromuscular junction defects and aberrant motor neuron development in knockout zebrafish (Lotti et al., 2012). Also, it has been reported that in TMEM41B-knockout HeLa cells there is an inhibition of Zika virus replication (Scaturro et al., 2018). Tmem41b has also been identified as a host cell factor for SARS-CoV-2 (Schneider et al., 2020). Tmem41b is the only common host cell factor identified for flaviviruses and coronaviruses and is the only autophagy-related protein identified as a viral host factor (Hoffmann et al., 2020).\n\nAdditionally, Tmem41b has been shown to be essential for mouse embryonic development: homozygous knockout mice embryos suffer early termination of their development after 7–8 weeks (Van Alstyne et al., 2018). Tmem41b is a structurally uncharacterised 291-residue protein found in the endoplasmic reticulum (ER) localising at the mitochondria-associated ER membranes (Moretti et al., 2018). Disruption of the PF09335 domain by various residue substitutions (Tábara et al., 2019) or its removal (Morita et al., 2018) results in inhibition of autophagosome formation and impaired lipid mobilisation in human embryonic kidney (HEK) cells.\n\nTmem41b homologues, hereafter referred to as VTT proteins (Morita et al., 2019), are present in all domains of life (Keller & Schneider, 2013). The Pfam PF09335 domain was first identified in the Saccharomyces cerevisiae protein Tvp38 (Inadome et al., 2007), and the authors concluded that Tvp38 associates with the tSNAREs in Tlg2-containing compartments, suggesting a role in membrane transport. Investigations into the bacterial and archaeal prevalence of these proteins showed that 90% of bacterial species and 70% of archaeal species encoded proteins with the PF09335 domain (Doerrler et al., 2013). Bacterial and archaeal PF09335-containing proteins are collectively known as the Death Effector Domain A (DedA) family (Doerrler et al., 2013). Detailed studies of the Escherichia coli DedA proteins have indicated that there are eight E. coli representatives of the DedA family (YqjA, YghB, YabI, Yoh, DedA, YdjX, YdjZ, and YqaA) with overlapping functions (Doerrler et al., 2013; Keller & Schneider, 2013), with Ydjx and Ydjz being the most closely related to human Tmem41b in terms of sequence similarity (Doerrler et al., 2013). Phenotypically, DedA knock-out E. coli cells display increased temperature sensitivity, cell division defects, activation envelope stress pathways, compromised proton motive force, sensitivity to alkaline pH and increased antibiotic susceptibility (Doerrler et al., 2013; Keller et al., 2014). As E. coli expresses multiple DedA homologues, the redundancy protects the cells from the phenotypical effects of single or multiple knock-outs as long as at least one DedA is expressed (Kumar & Doerrler, 2014). Borrelia burgdorferi contains only one DedA protein in its genome and knockout cells display the same phenotype as the E. coli knockout strains. Interestingly, E. coli knockout cells can be rescued with the B. burgdorferi homologue that shows only 19% sequence identity with YdjA. Attempts to rescue B. burgdorferi DedA knockout cells with E. coli homologues have resulted in more complex observations, with different homologues rescuing different phenotypes (Doerrler et al., 2013). The functions of DedA have also been studied in the opportunistic pathogen Pseudomonas aeruginosa where it was concluded that DedA proteins are required for its low antibiotic susceptibility. P. aeruginosa DedA is able to rescue E.coli DedA knockout cells (Justice et al., 2016).\n\nUntil the structure of poorly characterised protein families such as Pfam family PF09335 can be elucidated experimentally, ab initio protein modelling can be used to predict a fold allowing for structure-based function inferences (Rigden et al., 2017). Such methods have made significant strides recently due to the availability of contact predictions (Kinch et al., 2016). Prediction of residue-residue contacts relies on the fact that each pair of contacting residues covaries during evolution. The process of co-variation occurs as the properties of the two residues complement each other in order to maintain structural integrity of that local region and, consequently, its original functionality. Therefore, if one residue from the pair is replaced, the other must also change to compensate the physical chemical variation and hence preserve the original structure (Lapedes et al., 1999). The link between two residues can be then reliably detected in multiple sequence alignments by using direct coupling analysis (Morcos et al., 2011) as well as machine learning algorithms (Wu et al., 2020). The predicted contacts can be used for a range of analyses such as the identification of domain boundaries (Rigden, 2002; Simkovic et al., 2017a), but their main application is for contact-based modelling methods which can address larger targets than conventional fragment-assembly-based ab initio methods (Yang et al., 2020).\n\nIn the current study, we utilised state of the art methods to make structural predictions for two prominent members of the Pfam family PF09335 (Tmem41b and Yqja) by exploiting data derived from sequence, evolutionary covariance and ab initio modelling. We are able to predict that PF09335 homologues (VTT proteins) contain re-entrant loops (stretches of protein that enter the bilayer but exit on the same side of the membrane) as well as a pseudo-inverted repeat topology. The predicted presence of both of these structural features strongly suggests that VTT proteins are secondary active transporters for an uncharacterised substrate.\n\n\nMethods\n\nSearches using the sequences of VTT domain proteins Tmem41b, Yqja, Ydjx, Ydjz, Tvp38 and Mt2055 were made against the Pfam-A_v32.0 (RRID:SCR_004726) (El-Gebali et al., 2019) database using the HHPred (RRID:SCR_010276) v3.0 server (Zimmermann et al., 2018) with default parameters (-p 20 -Z 10000 -loc -z 1 -b 1 -B 10000 -ssm 2 -sc 1 -seq 1 -dbstrlen 10000 -norealign -maxres 32000 -contxt /cluster/toolkit/production/bioprogs/tools/hh-suite-build-new/data/context_data.crf) and eight iterations for MSA generation in the HHblits (Remmert et al., 2012) stage.\n\nThe DeepMetapsicov v1.0 server (Kandathil et al., 2019) was used to generate contact predictions with ConKit v0.12 (Simkovic et al., 2017b) utilised to visualise the contact maps. ConPlot (RRID:SCR_019216) was used to overlay additional prediction data (Sánchez Rodríguez et al., unpublished work).\n\nTransmembrane helical topology predictions were obtained from the Topcons server (Tsirigos et al., 2015). Secondary structure predictions were made employing a local installation of PSIPRED (RRID:SCR_010246) v4.0 (McGuffin et al., 2000). ConKit was also used to predict and visualise potential structural domain boundaries (Rigden, 2002; Simkovic et al., 2017a). Residue analysis of putative amphipathic regions were performed using HELIQUEST (Gautier et al., 2008) to determine the presence, direction and magnitude of any hydrophobic moment. Residue conservation was determined using the Consurf server (Ashkenazy et al., 2016).\n\nA library of re-entrant loop sequences together with the putative re-entrant loop sequences from the query proteins were clustered to establish any visible relationships of the sequences. The library was built by obtaining a non-redundant set of 56 re-entrant helix sequences by first retrieving all 714 TM proteins that contain at least one re-entrant loop from the PDBTM (RRID:SCR_011962) (Kozma et al., 2013) and removing redundancy with a 40% identity threshold. The resulting 127 protein structures were split into their component chains, eliminating any chain lacking a re-entrant loop. The subsequent set of 188 unique re-entrant loop sequences were then filtered removing any sequences of less than 10 residues and more than 20, thereby ensuring the collection of sequences conformed to the length of typical (Yan & Luo, 2010) re-entrant loops. The remaining 56 sequences were clustered, supplemented by candidate re-entrant sequences from the proteins studied here. Clustering was performed using CLANS v1.0 (Frickey & Lupas, 2004) with the BLAST results (p-value cut-off threshold of 0.1) (Altschul et al., 1997) used to calculate strengths of similarity.\n\nAb initio models were built using the trRosetta (Yang et al., 2020) server with default settings. Conservation was mapped on to the models using the ConSurf server (Ashkenazy et al., 2016). Visualisation of models was achieved using PyMOL (RRID:SCR_000305) v2.3.0 (DeLano, 2002).\n\nDali (RRID:SCR_013433) v4.0 (Holm & Laakso, 2016) was used to structurally align the output models and to query against the PDBTM (Kozma et al., 2013).\n\nAn earlier version of this article can be found on bioRxiv (doi: https://doi.org/10.1101/2020.06.27.174763)\n\n\nResults and discussion\n\nHHpred (Zimmermann et al., 2018) was used to screen a selection of VTT proteins against the Pfam database (El-Gebali et al., 2019). Hits were observed in the same region against both PF09335 and the Pfam domain PF06695 (‘Sm_multidrug_ex’) which is strongly indicative of homology: a probability of 99.4% with an E-value of 9E-17 for the PF09335 hit and 98.3% and 2E-10 respectively for PF06695. A HHpred search against the Pfam database using a member of PF06695 - the short archaeal sequence Mt2055 (UniProt code W9DY28) (Apweiler et al., 2004) - returned similar results (Table 1). The Mt2055 sequence originates from the unpublished draft genome of the archaebacterium Methanolobus tindarius DSM 2278. For many of the subsequent analyses, the shorter archaeal sequence was used initially but the clear homology among this set of proteins means that inferences can be drawn across the group.\n\nThere are no known experimental protein structures representing PF09335 or PF06695, but both Gremlin and DMPfold have constructed ab initio models for these Pfam domains (Greener et al., 2019; Ovchinnikov et al., 2017).\n\nAnalysis of the HHpred results obtained for the archaeal protein Mt2055 revealed the presence of additional hits for both PF06695 and PF09335 Pfam domains, in which the C-terminal half of the domains aligned with the N-terminal half of the Archaea protein. For example, residues 1-69 of the archaeal protein aligned with residues 52-117 of the Pfam PF09335 profile with a probability of 74.15%. Interestingly, contact density analysis (Rigden, 2002; Sadowski, 2013) supported the existence of a domain boundary around residue 60, in broad agreement with the HHpred results (Figure 1). Both the HHpred and contact density results therefore pointed to a specific domain structure being present.\n\n(a) Contact density profile constructed by ConKit (Simkovic et al., 2017b) utilising DeepMetaPSICOV contact prediction. Solid black line represents contact density and dotted red lines mark density minima corresponding to possible domain boundaries. (b) HHalign alignments for the N-terminal and C-terminal Mt2055 halves, formatted using Jalview (Waterhouse et al., 2009) and coloured according to the ClustalX scheme. Red bars represent helical secondary structure. (c) Maps of predicted contacts generated by DeepMetaPSICOV and plotted using ConKit; left is N-terminal half (residues 1-84) and right is C-terminal half (residues 85-168). Black points represent predicted intramolecular contacts.\n\nWhen the Mt2055 sequence was split at residue 60-61, the resulting N-terminal region of 60 residues and the C-terminal section of 79 residues could be aligned using HHalign (Soding, 2005) with a 78% probability and an E-value of 1.9E-3. Examination of the map of predicted contacts for Mt2055 reveals features that are present in both the N- and C-terminal halves of the protein (Figure 1c). Taken together, these data strongly support the existence of a tandem repeat within the Mt2055 protein and hence across the PF06695 and PF09335 protein families.\n\nInterestingly, however, an equivalent sequence analysis with HHpred of other PF09335 homologues including Tmem41b itself does not reveal a repeat. However, inspection of their corresponding predicted contact maps does reveal features repeated when N- and C-halves of the protein are compared (Figure 2). Apparently, evolutionary divergence has removed all trace of the repeat sequence signal in bacterial and eukaryotic proteins, although the feature remains visible by evolutionary covariance analysis.\n\nThe highlighted areas represent repeat units that have been revealed through evolutionary covariance analysis.\n\nSeveral authors have deposited structures of uncharacterised Pfam families in databases (El-Gebali et al., 2019); however, Pfam domain boundaries for PF09335/PF06695, which define the limits of these previous modelling exercises, do not reflect the conserved structural domain that we predict. Given the fact that the available ab initio models were inconsistent with the transmembrane helix, secondary structure and contact predictions (data not shown), we constructed our own models of Mt2055 as well as Tmem41b and Yqja with trRosetta.\n\nThe Mt2055, Tmem41b and Yqja models had estimated TM scores from the trRosetta server of 0.633, 0.624 and 0.635 respectively, suggesting that they were likely to have captured the native fold of the family. All-against-all pairwise structural superposition of the models with DALI gave a mean Z-score of 11.9 confirming their strong similarity. We also used satisfaction of predicted contacts to validate the models (Figure 3) (Simkovic et al., 2017a). This showed that 80% of the top L predicted contacts (where L is the length of the protein) are satisfied by the model contacts for both Mt2055 and Yqja and a value of 60% was achieved for Tmem41b suggestive of good quality models (de Oliveira et al., 2017).\n\n(a) trRosetta model of MT2055 - amphipathic helix (green) and a re-entrant loop (orange) packed with a TM helix (red) (b) Superposition of DMP predicted contact map for Mt2055 and contacts from the Mt2055 model. Black points are matching contacts, red are mismatches and grey are contacts predicted but not present in the model. Diagonal is a visual representation of transmembrane helix and secondary structure prediction – central diagonal is the visualisation of the TopCons transmembrane prediction (orange being a TM helix) and the outer diagonals are the visual representation of the PsiPred secondary structure prediction (pink – alpha helix and yellow – coil). Red boxes highlight the re-entrant loop and TM helix packing. c) trRosetta model of Tmem41b only showing the conserved structural domain (residues 39-217) d) trRosetta model of Yqja only showing the conserved structural domain (residues 14-176). e) Proposed topology for (extended) VTT domain.\n\nThe models (Figure 3) contained interesting features: two inversely symmetrical repeated units each possessing an amphipathic helix (green) and a re-entrant loop (orange) packed with a TM helix (red).\n\nThe presence of a re-entrant loop packed against each TM helix can also be seen on predicted contact maps for these proteins (Figure 3b). Interestingly, each of the re-entrant helices is predicted as a single transmembrane region in the TopCons predictions (see the diagonal of Figure 3b) with a two-residue region of coil in the centre. Such a prediction would more obviously be treated as indicative of some kind of kink in the helix (Law et al., 2016) but the explanation here is these regions form re-entrant helices. Similar contact map features, indicative of re-entrant loops packing against TM helices, can be seen clearly on the contact maps of other VTT proteins (data not shown).\n\nThe analysis was performed by HELIQUEST (Gautier et al., 2008) which constructed helical wheel diagrams and provided a quantitative measure of the hydrophobic moment for the region being analysed (Figure 4).\n\nHydrophobic residues are shown in yellow, serine and threonine in purple, basic residues in dark blue, acidic residues in red, asparagine and glutamine in pink, alanine and glycine in grey, histidine in light blue and proline in green circles. Arrows represent direction and magnitude of the hydrophobic moment and residue marked with ‘N’ is the N-terminal end of the putative amphipathic helix with the residue marked ‘C’ being the C-terminal end. (a) Mt2055 putative amphipathic helix 1 (hydrophobic moment of 0.298). (b) Mt2055 putative amphipathic helix 2 (hydrophobic moment of 0.546). (c) Tmem41b putative amphipathic helix 1 (hydrophobic moment of 0.471). (d) Tmem41b putative amphipathic helix 2 (hydrophobic moment of 0.420). (e). Yqja putative amphipathic helix 1 (hydrophobic moment of 0.295). (f) Yqja putative amphipathic helix 2 (hydrophobic moment of 0.396).\n\nMapping conservation onto the models, using the Consurf server, indicates that the re-entrant loops are highly conserved and therefore likely to be functionally and/or structurally important (Figure 5).\n\ntrRosetta models with Consurf conservation mapping for (a) Mt2055 (b) Tmem41b (c) Yqja. Conservation is shown as a spectrum from blue (highly conserved) to red (not conserved).\n\nThe presence of re-entrant loops and the high density of conserved residues within them caused us to examine experimentally characterised re-entrant loops in the PDBTM database. A total of 56 non-redundant re-entrant helices were identified (see Methods). All 56 were clustered with the putative re-entrant loops from Mt2055 and four PF09335 homologues (Tmem41b, Tvp38, Ydjx and Ydjz) using relative E-values derived from an all-against-all BLAST run in CLANS (Frickey & Lupas, 2004) with a 0.1 p-value cut-off. The largest cluster contained 14 sequences, of which four were putative re-entrant sequences from the query proteins (Mt2055 C-terminal re-entrant, Ydjx C-terminal re-entrant, Ydjz N-terminal re-entrant & Ydjz C-terminal re-entrant), seven (3org, 5tqq, 3nd0, 3det and 6coy) were re-entrant loop sequences from Cl-/H+ antiporters, one was from a boron exchanger (5l25), one from an electron transporter (2n4x) [albeit classified as a member of the lysine exporter superfamily (Saier et al., 2016)] and one from a mechanogated channel (5z10).\n\nAnalysis of the Cl-/H+ antiporter structures show that they contain a similar inverted repeat as we infer for the VTT homologues, resulting in pseudo-2-fold axis of symmetry running along the membrane (Duran & Meiler, 2013). Again similarly, the Cl-/H+ antiporter 3orgA also contains the amphipathic helices on the N-terminal side of the re-entrant loops. The fact that the presence of the amphipathic helices is restricted only to 3orgA and not found in all homologues suggest that these features are not essential for function (Figure 5).\n\nThe presence of re-entrant loops in a transmembrane protein strongly indicates a transporter or pore functionality since this structural feature has, hitherto, only been found in proteins of this kind (Yan & Luo, 2010). The structural similarities between the VTT proteins and the Cl-/H+ antiporters raise the possibility that the families studied here are, in fact, unsuspected distant homologues having this putative pore feature in common. In that regard it is relevant to recall a hypothesis that DedA proteins are H+ antiporters resulting from SDM experiments (Justice et al., 2016).\n\nA recent study has identified key residues (Figure 6) in the E. coli DedA protein Yqja that, when replaced in site directed mutagenesis experiments, resulted in decreased proton motive force across the E. coli inner membrane (Panta et al., 2019). Highlighting the essential residues (E39, D51, R130 and R136) on the Yqja model show that they come together in three-dimensional space with the N-terminal side of the first re-entrant possessing E39 and the C-terminal side possessing D51. R130 and R136 are similarly positioned on the second re-entrant loop (Figure 7). Re-entrant loops are known to form pores and here we have two proton-titratable residues (E39, D51) in close proximity to essential basic residues (R130 and R136) within a putative pore. This three-dimensional arrangement of key residues could serve a role in the coupling of the protonation status with the binding of a yet to be characterised substrate as is postulated for the multi-drug H+ antiporter MdfA (Heng et al., 2015) where these same residues are located inside a central cavity.\n\n(a) Left - Predicted Contact map with repeating units highlighted in yellow boxes, contact map signature of re-entrant loop packed with TM helix in red boxes.; Right - The Experimental Contact map obtained from the PDB structure with repeating units highlighted in yellow boxes, contact map signature of re-entrant loop packed with TM helix in red boxes. (b) Actual 3orgA topology; grey: TM Helices that are additional to the core; red: TM helices contributing to the formation of the core; orange; re-entrant loops contributing to the formation of the core; green: amphipathic helices contributing to the formation of the core. (c) The 2-fold pseudo symmetry of the amphipathic/re-entrant loop/TM helix core inverted repeat structure of 3orgA with membrane positions shown as grey planes obtained from PDBTM.\n\n\nConclusions\n\nThis study demonstrates how covariance prediction data have multiple roles in modern structural bioinformatics: not just by acting as restraints for model making and serving for validation of the final models but by predicting domain boundaries and revealing the presence of cryptic internal repeats not evidenced by sequence analysis. Furthermore, we characterised a contact map feature characteristic of a re-entrant helix which may in future allow detection of this feature in other protein families.\n\nSequence, co-variance and ab initio modelling analyses show that the Pfam PF09335 and PF06695 domains are distantly homologous. These domains contain a structural core composed of a pseudo-inverse repeat of an amphipathic helix, a re-entrant loop and a TM helix. All PF09335 homologues contain this central core with additional TM- helices flanking either side.\n\nQuerying the models against the PDB using Dali did not yield any significant hits. However, analysis of the prediction data revealed two features of VTT proteins that independently suggest that they are secondary transporters: both an inverted repeat architecture and the presence of a re-entrant loop, which are both independently and strongly associated with transporter function (Duran & Meiler, 2013; Yan & Luo, 2010). Additionally, the fact that VTT proteins show structural similarities with H+ antiporters indicate that these proteins may also couple substrate transport with an opposing H+ current. Indeed, the Yqja homologue also contains strategically placed residues known to be involved in H+ antiporter activity. The ab initio models show that the essential residues come together in the region that would be buried in the membrane potentially forming a substrate chamber consistent with the transport of a specific substrate. Further research needs to be carried out to determine what this substrate is and confirm the mechanism of transport.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "References\n\nAlmén MS, Nordström KJ, Fredriksson R, et al.: Mapping the human membrane proteome: a majority of the human membrane proteins can be classified according to function and evolutionary origin. BMC Biol. 2009; 7: 50. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAltschul S, Madden TL, Schäffer AA, et al.: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997; 25(17): 3389–3402. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "75807", "date": "17 Dec 2020", "name": "Pradip Panta", "expertise": [ "Reviewer Expertise Bacterial genetics with interests in membrane proteins and antibiotic resistance mechanisms." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis work describes the computational structural modeling of a conserved membrane protein family that includes human TMEM41B, a protein with a number of reported functions. Membrane proteins are poorly represented in the structural database and computational methods are increasingly valuable for understanding structure and function. Here, they use a method using evolutionary amino acid contact co-variation to predict a structure that supports a proposed function as a proton dependent antiporter. While I am not fluent in the computational methods used, their prediction do align with published experimental work. The manuscript is well written and informative, but with a number of factual errors. I also suggest additional citations.\nI would like to begin with nomenclature. I received an email from Dr. Noburo Mizushima several months ago. He has published work on the TMEM41B protein. Also included on the email was Lucy Forrest, Dirk Schneider, and Rebecca Keller. It was Dr. Mizushima’s suggestion to name this protein family the \"DedA superfamily\" that includes both prokaryotic and eukaryotic proteins (DedA, VMP, and TMEM41 families). Accordingly, the shared domain will be called \"DedA domain\" and \"VTT\" domain would no longer be used. All recipients of this email agreed to using this nomenclature moving forward. Therefore, to avoid confusion, I would like the authors to adopt this nomenclature. I can forward the email upon request.\nSince the manuscript contains no line numbers, I will list the suggested corrections by paragraph: Introduction: Paragraph 1: Formally, “membrane proteins” also include various lipid-modified proteins of both prokaryotes and eukaryotes in addition to integral and peripheral membrane proteins.\nParagraph 4: “DedA” does not stand for “death effector domain”. It was named in a 1987 paper1. See page 12213 of that article. I would like to see this article cited as well for historical purposes.\nThe sentence that begins with “Phenotypically, DedA knockout E. coli…” should instead read “Phenotypically, E. coli lacking both yqjA and yghB (encoding proteins with 60% amino acid identity and partially overlapping functions)….\" This paragraph should also cite2.\n\nThe sentence that reads “As E. coli expresses multiple DedA homologues, the redundancy protects the cells from the phenotypical effects of single or multiple knock-outs as long as at least one DedA is expressed” should read “As E. coli expresses multiple DedA homologues, lethal effects are not observed as long as at least one DedA is expressed”. Cite the following article 3.\nYou may also point out that the sole DedA family gene in Borrelia burgdorferi is indeed essential4.\nYqjA is misspelled “YdjA”\nThe sentence “Attempts to rescue….” Should be removed, as it does not make sense.\nThe final sentence about Pseudomonas cites a non-peer reviewed proceeding abstract. I would like all citations to “Justice et al. 2016” removed from this article. This sentence can be replaced with the equally effective “The functions of DedA have also been studied in the pathogen Burkholderia thailandensis where one family member was found to be required for resistance to polymyxin”5.\nParagraph 6: “YqjA” is spelled “Yqja” here and throughout the manuscript and should be corrected. This includes in Table 1.\nMethods:\nParagraph 1: Please spell “Ydjx” and other bacterial proteins as “YdjX” with the final letter capitalized.\nResults and discussion: Paragraph 5: first sentence, remove “however”.\nParagraph 14: “A possible role for VTT proteins” final sentence remove “Justice et al.” and instead cite 6,7. Also, in this sentence, define “SDM” as “site directed mutagenesis”.\nParagraph 15, first sentence. This statement is incorrect. Mutation of D51, E39, R130 or R136 in YqjA resulted in properly folded (membrane localized) but nonfunctional proteins unable to complement alkaline pH sensitivity of E. coli YqjA mutant and antibiotic sensitivity of YqjA/YghB double mutant.\nFinally, another interesting example of a membrane protein antiporter with re-entrant helices is the undecaprenyl pyrophosphate phosphatase UppP. It is up the authors if they would like to cite these articles 8,9.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6431", "date": "25 Mar 2021", "name": "Daniel Rigden", "role": "Author Response", "response": "I would like to begin with nomenclature. I received an email from Dr. Noburo Mizushima several months ago. He has published work on the TMEM41B protein. Also included on the email was Lucy Forrest, Dirk Schneider, and Rebecca Keller. It was Dr. Mizushima’s suggestion to name this protein family the \"DedA superfamily\" that includes both prokaryotic and eukaryotic proteins (DedA, VMP, and TMEM41 families). Accordingly, the shared domain will be called \"DedA domain\" and \"VTT\" domain would no longer be used. All recipients of this email agreed to using this nomenclature moving forward. Therefore, to avoid confusion, I would like the authors to adopt this nomenclature. I can forward the email upon request. Thanks very much for this helpful suggestion. We have changed the naming in the manuscript throughout to “DedA superfamily” Introduction: Paragraph 1: Formally, “membrane proteins” also include various lipid-modified proteins of both prokaryotes and eukaryotes in addition to integral and peripheral membrane proteins. Thank you, this omission has been rectified; ‘Membrane proteins can be grouped according to their interaction with various cell membranes: integral membrane proteins (IMPs) are permanently anchored whereas peripheral membrane proteins transiently adhere to cell membranes. IMPs that span the membrane are known as transmembrane proteins (TMEMs) as opposed to IMPs that adhere to one side of the membrane ( Fowler & Coveney, 2006). Membrane proteins also include various lipid-modified proteins (Resh, 2016).’ Paragraph 4: “DedA” does not stand for “death effector domain”. It was named in a 1987 paper1. See page 12213 of that article. I would like to see this article cited as well for historical purposes. Your clarification on this nomenclature is appreciated. We have amended the manuscript to reflect this error and included the reference for its historical importance. The sentence that begins with “Phenotypically, DedA knockout E. coli…” should instead read “Phenotypically, E. coli lacking both yqjA and yghB (encoding proteins with 60% amino acid identity and partially overlapping functions)….\" This paragraph should also cite2. The sentence that reads “As E. coli expresses multiple DedA homologues, the redundancy protects the cells from the phenotypical effects of single or multiple knock-outs as long as at least one DedA is expressed” should read “As E. coli expresses multiple DedA homologues, lethal effects are not observed as long as at least one DedA is expressed”. Cite the following article 3. Paragraph 14: “A possible role for VTT proteins” final sentence remove “Justice et al.” and instead cite 6,7. The additional references for previous experimental studies of the DedA proteins that you have suggested have been added to the manuscript. Thanks for your suggestions which certainly make the manuscript more comprehensively cite previous studies. You may also point out that the sole DedA family gene in Borrelia burgdorferi is indeed essential4. The final sentence about Pseudomonas cites a non-peer reviewed proceeding abstract. I would like all citations to “Justice et al. 2016” removed from this article. This sentence can be replaced with the equally effective “The functions of DedA have also been studied in the pathogen Burkholderia thailandensis where one family member was found to be required for resistance to polymyxin”5. Thanks for these helpful suggestions. The inclusion of these points brings more clarity to the paragraph in question; “Borrelia burgdorferi contains only one DedA protein in its genome and knockout cells display the same phenotype as the E. coli knockout strains. The B. burgdorferi homologue is indeed essential (Liang et al., 2010). Interestingly, E. coli knockout cells can be rescued with the B. burgdorferi homologue that shows only 19% sequence identity with YqjA. The functions of DedA have also been studied in the pathogen Burkholderia thailandensis where one family member was found to be required for resistance to polymyxin (Panta et al., 2019).” YqjA is misspelled “YdjA” Paragraph 6: “YqjA” is spelled “Yqja” here and throughout the manuscript and should be corrected. This includes in Table 1. Paragraph 1: Please spell “Ydjx” and other bacterial proteins as “YdjX” with the final letter capitalized. Corrections made. Thank you for pointing out these important errors. The sentence “Attempts to rescue….” Should be removed, as it does not make sense. Sentence removed, thank you. Results and discussion: Paragraph 5: first sentence, remove “however”. Amendment made, thank you. Also, in this sentence, define “SDM” as “site directed mutagenesis”. Amendment made, thank you. Paragraph 15, first sentence. This statement is incorrect. Mutation of D51, E39, R130 or R136 in YqjA resulted in properly folded (membrane localized) but nonfunctional proteins unable to complement alkaline pH sensitivity of E. coli YqjA mutant and antibiotic sensitivity of YqjA/YghB double mutant. Thank you for pointing this out to us. We can see our description of the results of the study was incorrect. This has been amended with your direction; “A recent study has identified key residues ( Figure 8) in the E. coli DedA protein YqjA that, when replaced in site directed mutagenesis experiments, resulted in properly folded (membrane localized) but non-functional proteins unable to complement alkaline pH sensitivity of E. coli YqjA mutant and antibiotic sensitivity of YqjA/YghB double mutant ( Panta et al., 2019).” Finally, another interesting example of a membrane protein antiporter with re-entrant helices is the undecaprenyl pyrophosphate phosphatase UppP. It is up the authors if they would like to cite these articles 8,9. Yes, this is interesting; for the revision of the manuscript we re-implemented the re-entrant loop screen against the PDBTM. We found that 6cb2 (UppP) re-entrant loop structures were structurally very similar to the re-entrant models for the DedA domains." } ] }, { "id": "75806", "date": "07 Jan 2021", "name": "Laszlo Dobson", "expertise": [ "Reviewer Expertise Topology and structure prediction of transmembrane proteins." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this manuscript Mesdaghi et al. describe the in silico structure modeling of three homologous integral membrane proteins Mt2055, Yqja and human Tmem41b. Structure determination of transmembrane proteins lacks behind globular ones for several reasons, giving space for computational tools. The proper use of these tools may unveil important structural aspects of transmembrane proteins but interpretation of results of such analysis should be done carefully. While the generated models in the manuscript are interesting and might be fully or partly true, the sequence analysis and interpretation of the results are problematic.\nMajor: - The authors should be more specific about the exact boundaries of Pfam domains in different proteins as well as the sequence relations of proteins presented in Table 1. Please provide multiple sequence alignment for these proteins indicating the localization of the two pfam domains and the proposed re-entrant loops/transmembrane regions in the sequences.\n- The authors propose Mt2055 contains a tandem repeat and suggest the duplication is present in Tmem41b and Yqja structure as well even if it is undetectable from sequence analysis. The proposed domain boundary in Figure1a and arguments for tandem duplication does not seem convincing. The e-value of 1.9E-3 is quite large for the alignment. The authors should rule out that results in their paper may occur purely by chance. Please test the statistical significance of this value by generating pairwise alignments of transmembrane regions of unrelated transmembrane proteins with similar length. Moreover, contact maps for Mt2055 and Tmem41b were generated from the same multiple alignment, and therefore they must be identical/similar. Thus the similarities does not prove the tandem duplication occurred in Tmem41b too.\n- Structure modeling of membrane proteins is somewhat different from globular ones for several reasons. It is highly recommended to use specific software for this task or argue why used a non-specific one. On one hand, in general, topology prediction is more accurate than structure modeling and should be used as an input to aid the modeling. The reviewer is not sure the result of a standard ab initio structure modeling program is sufficient to question topology prediction results. On the other hand, topology prediction results are different for Tmem41b (6 TM helix) and Mt2055 (4 TM helix). Notably, other consensus topology method (CCTOP) have a similar result for Mt2055 (4 helix), but different for Tmem41b (6 helix). Using a third method (Octopus) a re-entrant loop is predicted. The authors should elaborate on such results instead of picking one method and running it on only one of the sequences.\n- Authors state: “For many of the subsequent analyses, the shorter archaeal sequence was used initially but the clear homology among this set of proteins means that inferences can be drawn across the group.” - Please provide the used multiple sequence alignment with pairwise similarities to support this statement.\n- It is not clear how helical wheels and hydrophobic moments support the manuscript - please provide a better description or omit these results.\n- Problems/Validation of re-entrant loops:\nThe authors selected 56 sequence regions from PDBTM database and run an all-against-all Blast search and create clusters based on the search results. Since the sequence complexity of membrane regions are lowest than regions of globular proteins, the analysis should be repeated on randomly selected transmembrane segments. Please provide the list of the selected 56 re-entrant loops together with the results of the repeated analysis.\n\nAuthors state: “The presence of a re-entrant loop packed against each TM helix can also be seen on predicted contact maps for these proteins (Figure 3b).” Re-entrant loops cannot be seen on contact map, only parallel and anti-parallel structures. A similar contact map can be easily generated from 3 transmembrane helices (1 parallel pair and two anti-parallel ones).\n\nThe authors filtered removing any sequences of less than 10 residues and more than 20. Although the exact sequence localisation and length of the predicted re-entrant loop are not provided, the regions indicated as the “sign” of re-entrant loops on Figure 3b is larger than 20 residues and on the structures the orange regions contain 7 turns, thus the sequence length of them should be more than 20 residues (73.5=24.5).\n\nThe most serious one: As it can be seen on Fig7b-c, 3org contains additional helices that surround the interfacial helix - re-entrant loop - tm structure. Indeed the protein are dimer, where the dimer interface are formed by the re-entrant loops and the additional transmembrane helices surround this core. This arrangement ensure the lipid embedded structure is energetically stable. In the proposed model, re-entrant loops are not wrapped by other helices thus lipids may interact them. This is energetically unfavorable and does not prefer for the suggested function too. The validity of the model should be further investigate by molecular dynamic simulations of lipid embedded structures.\n- “The analysis was performed by HELIQUEST (Gautier et al., 2008) which constructed helical wheel diagrams and provided a quantitative measure of the hydrophobic moment for the region being analysed (Figure 4).” - This sentence and Figure 4 are pointless, containing data not used in the validation of the results.\nMinor:\nAbstract/Results: “The results from the structural bioinformatics analysis of Tmem41b and its homologues showed that they contain a tandem repeat that is clearly visible in evolutionary covariance data but much less so by sequence analysis.” - As I showed above, this statement might not be true. Moreover evolutionary covariance data is the results of sequence analysis, so this sentence is void of sense. Please rephrase.\n\nIntroduction: “there are eight E. coli representatives of the DedA family (YqjA, YghB, YabI, Yoh, DedA, YdjX, YdjZ, and YqaA)” - Character D is missing in Yoh.\n\nIntroduction: “In the current study, we utilised state of the art methods to make structural predictions for two prominent members of the Pfam family PF09335 (Tmem41b and Yqja) by exploiting data derived from sequence, evolutionary covariance and ab initio modelling.” - The most part of the manuscript deal with the sequence analysis of Mt2055, please rephrase this sentence in order to mirror this fact.\n\n\"Interestingly, each of the re-entrant helices is predicted as a single transmembrane region in the TopCons predictions (see the diagonal of Figure 3b) with a two-residue region of coil in the centre.\" - TOPCONS does not predict coils and such details cannot be seen on the figure - please clarify this sentence.\n\nThe authors should provide the generated PDB files as Supplementary Material.\n\nContact map on Figure 7a left is the same that on right (numbering, dots). They should be different if one based on prediction and the other based on experimental data.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "6435", "date": "25 Mar 2021", "name": "Daniel Rigden", "role": "Author Response", "response": "Major: - The authors should be more specific about the exact boundaries of Pfam domains in different proteins as well as the sequence relations of proteins presented in Table 1. Please provide multiple sequence alignment for these proteins indicating the localization of the two pfam domains and the proposed re-entrant loops/transmembrane regions in the sequences. Yes agreed, we agree that a MSA is useful. An MSA generated using PSI/TM-COFFEE has been added as Figure 1 to the manuscript. The Pfam domains in questions well as the putative re-entrant loops for the modelled proteins have been highlighted to illustrate their relative positions. - The authors propose Mt2055 contains a tandem repeat and suggest the duplication is present in Tmem41b and Yqja structure as well even if it is undetectable from sequence analysis. The proposed domain boundary in Figure1a and arguments for tandem duplication does not seem convincing. The e-value of 1.9E-3 is quite large for the alignment. The authors should rule out that results in their paper may occur purely by chance. Please test the statistical significance of this value by generating pairwise alignments of transmembrane regions of unrelated transmembrane proteins with similar length. Utilisation of HHalign does result in an e-value of 1.9E-3 which on its own is not compelling. However, as highlighted, HHalign also expressed a probability score (a measure of statistical significance) of 78% which the software developers argue is a better indicator of significance than the e-value alone. Additionally (as explained earlier in the text) ‘Analysis of the HHpred results (against Pfam database) obtained for the archaeal protein Mt2055 revealed the presence of additional secondary hits for both PF06695 and PF09335 Pfam domains, in which the C-terminal half of the domains aligned with the N-terminal half of the Archaea protein. For example, residues 1-69 of the archaeal protein aligned with residues 52-117 of the Pfam PF09335 profile with a probability of 74.15%.‘ It is important to remember that generating and scoring alignments with unrelated TM proteins is an intrinsic part of the database search. We have estimated that in Pfam there are currently around 1377 Pfam domains containing two or more TM-helices. This is based on analysis of the Phobius predictions that are part of the current 33.1 release. In the search above, only three of these domains - PF08566, PF09835, PF13571 - scored comparably (probabilities of 73.3-76.6%) with the secondary hits for PF06695 and PF09335 . These results clearly place the secondary PF06695 and PF09335 matches at the extreme end of the score distribution for TM-helical Pfam domains, supporting their significance. Arguably the above findings alone do not provide absolutely conclusive evidence of the presence of a repeat. However, reinforcing these findings we have the repeat that is revealed by the plotting of the predicted contacts and, consequently, the inverse repeat that is witnessed by the modelling. Moreover, contact maps for Mt2055 and Tmem41b were generated from the same multiple alignment, and therefore they must be identical/similar. Thus the similarities does not prove the tandem duplication occurred in Tmem41b too. Interesting point: however, we can confirm that the MSAs used to generate the contacts maps for Mt2055 and Tmem41b were not identical. The MSAs constructed by DMP were constructed independently using HHblits against the Uniprot database. The manuscript used the predicted contacts from the server and the MSAs generated to make the contact predictions are not made available to download with the results. However, performing the contact prediction locally and utilising the same HHblits settings as the DMP server generates MSAs with 5000 sequences for each of the query proteins. The predicted contact maps are very similar to those presented in the paper yet analysis reveals that the MSAs had only 1010 sequences in common. - Structure modeling of membrane proteins is somewhat different from globular ones for several reasons. It is highly recommended to use specific software for this task or argue why used a non-specific one. At the beginning of this project we had similar thoughts to you, therefore initially Rosetta membrane was utilised to build the models. However, the membrane protocol ‘forced’ TM helices where it was later clear from contact map analysis that re-entrant loops should be present. Therefore, it was decided that contact restrained modelling software with proven success in regard to ab initio modelling of membrane proteins was used. Both DMPfold (local & server) as well as the trRosetta (server) models were constructed and similar folds were observed. We note that the DMPfold paper benchmarked using transmembrane protein as explicitly says it ‘works just as well for transmembrane proteins.’ (https://www.nature.com/articles/s41467-019-11994-0 ). The trRosetta method was benchmarked against CASP13 targets which included transmembrane proteins (https://onlinelibrary.wiley.com/doi/abs/10.1002/prot.25775). The use of these covariance-based methods for membrane proteins has a long history so the following citation has been included in the revised manuscript; Hopf, T. A., Colwell, L. J., Sheridan, R., Rost, B., Sander, C., & Marks, D. S. (2012). Three-dimensional structures of membrane proteins from genomic sequencing. Cell, 149(7), 1607–1621. On one hand, in general, topology prediction is more accurate than structure modelling and should be used as an input to aid the modelling. The reviewer is not sure the result of a standard ab initio structure modelling program is sufficient to question topology prediction results. On the other hand, topology prediction results are different for Tmem41b (6 TM helix) and Mt2055 (4 TM helix). Notably, other consensus topology method (CCTOP) have a similar result for Mt2055 (4 helix), but different for Tmem41b (6 helix). Using a third method (Octopus) a re-entrant loop is predicted. The authors should elaborate on such results instead of picking one method and running it on only one of the sequences. The different membrane topology prediction tools were used initially to predict the TMhelix boundaries for the query proteins. We observed the same between the results of the different methods as yourself. It was the variability of the topology predictions in addition to the contact map features that led to the conclusion that something other than straightforward TM helices is present in the Pfam domains in question. Indeed, TMHMM does show lower probability TMhelix predictions for the regions that the contact prediction and model making predict to be re-entrant loops. To investigate further, visual representations of the membrane topology from TopCons and the psipred secondary structure prediction were plotted along the diagonal of the contact prediction for the query proteins. This clearly highlights that the N and C halves of the predicted TM helices in question are making contact with each other (by a length of around 10 residues). Additionally, the secondary structure plot shows an interruption at the halfway point of the predicted TM helices which would account for the abrupt change in direction of helix in the membrane. Additionally, we have identified a crystal structure that is comparable in terms of size (293 residues) and has common structural features (inverted repeat with 2 re-entrant/TMhelix structures) to our query proteins; 6cb2. For this protein, the TopCons topology prediction was compared to the actual topology of the crystal structure. The above figure shows actual contacts for 6cb2 (black points) and a visual representation of the TopCons topology prediction (green -outside, red – TM helix, yellow-inside, yellow boxes are the re-entrant loop-TM-helix ‘signature’). Cross-referencing the first re-entrant contact map feature with the TopCons topology prediction it is clear that the TopCons topology must be wrong; the first TopCons predicted TM helix cannot be making contact with a region out-side of the membrane. Indeed, examination of the crystal structure reveals that the contact feature highlighted does in fact result from a re-entrant loop packed with a TMhelix . Furthermore, constructing ab initio models of 6cb2 using both trRosetta (server) and DMPfold (server) yielded models that correctly fold the re-entrant loop in question. Performing structural alignments of the ab initio models against the crystal structure using Dali (server) give Z scores of 35 (trRosetta) and 27.5 (DMPfold). These scores are leave no doubt that the correct fold has been modelled. The image below shows the crystal structure in green and the trRosetta model in magenta. The second image highlights the re-entrant feature that we are interested in. - Authors state: “For many of the subsequent analyses, the shorter archaeal sequence was used initially but the clear homology among this set of proteins means that inferences can be drawn across the group.” - Please provide the used multiple sequence alignment with pairwise similarities to support this statement. A multiple sequence alignment is now provided as Figure 1 and we note that all query sequences share the same Pfam domains so their homology is assured. - It is not clear how helical wheels and hydrophobic moments support the manuscript - please provide a better description or omit these results. - “The analysis was performed by HELIQUEST (Gautier et al., 2008) which constructed helical wheel diagrams and provided a quantitative measure of the hydrophobic moment for the region being analysed (Figure 4).” - This sentence and Figure 4 are pointless, containing data not used in the validation of the results. Yes, we are in agreement with you, the paragraph did seem out of place as well as unfinished. In response we have re-worked the paragraph in question providing more clarity and analysis for the amphipathic analysis of the queries; “In order to test for the presence of the amphipathic helices, an analysis of helical wheel diagrams for the fifteen residues preceding the putative re-entrant loops was performed with HELIQUEST ( Gautier et al., 2008). The quantitative measures of the hydrophobic moment for the regions being analysed (Figure 5) support that they are indeed amphipathic helices. The hydrophobic moments ranged from 0.298 to 0.546.” To clarify; from the helical wheel figures the amphipathic nature of the approximately 15 residues preceding the putative re-entrant loops is clear. The importance of this finding is explained in relation to the structural comparison with the Cl-/H+ anti-porter 3org which also possesses the same structural features that we predict for the DedA proteins. - Problems/Validation of re-entrant loops: • The authors selected 56 sequence regions from PDBTM database and run an all-against-all Blast search and create clusters based on the search results. Since the sequence complexity of membrane regions are lowest than regions of globular proteins, the analysis should be repeated on randomly selected transmembrane segments. Please provide the list of the selected 56 re-entrant loops together with the results of the repeated analysis. • The authors filtered removing any sequences of less than 10 residues and more than 20. Although the exact sequence localisation and length of the predicted re-entrant loop are not provided, the regions indicated as the “sign” of re-entrant loops on Figure 3b is larger than 20 residues and on the structures the orange regions contain 7 turns, thus the sequence length of them should be more than 20 residues (73.5=24.5). • The most serious one: As it can be seen on Fig7b-c, 3org contains additional helices that surround the interfacial helix - re-entrant loop - tm structure. Indeed the protein are dimer, where the dimer interface are formed by the re-entrant loops and the additional transmembrane helices surround this core. This arrangement ensure the lipid embedded structure is energetically stable. In the proposed model, re-entrant loops are not wrapped by other helices thus lipids may interact them. This is energetically unfavorable and does not prefer for the suggested function too. The validity of the model should be further investigate by molecular dynamic simulations of lipid embedded structures. Yes we are in agreement with you; the imposition of the re-entrant loop boundaries for the ab initio models were relatively arbitrary. Therefore, the re-entrant loop screen against the PDBTM has been completely re-implemented. Membrane boundaries for the models have now been predicted using the OMP server. These boundaries provide the lengths of the putative re-entrant loops. Consequently it is now recognised that the 20 residue ‘typical length’ of re-entrant loops may not be valid for the query models and the filtering of the larger loops for the clustering stage of this research had weak justification. The clustering Methods text now reads “A library of re-entrant loop pdb structures together with the putative re-entrant loop structures from the query protein models were clustered on their structural similarity. The library was built by obtaining a non-redundant (removing redundancy with a 40% sequence identity threshold) set of 125 chains from the PDBTM (RRID:SCR_011962) ( Kozma et al., 2013) that contain at least one re-entrant loop. As this investigation focuses on re-entrant loops that are immediately preceded by a TM helix that is packed against the loop, all re-entrant loops (boundaries defined by PDBTM) in addition to the preceding 30 residues were extracted. The resulting 193 library entries (https://figshare.com/articles/dataset/repository_zip/14055212), supplemented with the re-entrant loop features (defined by the OMP server (Lomize, Pogozheva, Joo, Mosberg, & Lomize, 2012) and accompanied by the preceding 30 residues ) from the ab initio modelling underwent an all-against-all structural alignment using a local installation of Dali v4.0 ( Holm & Laakso, 2016). The Z-scores for these alignments were then used for clustering with CLANS v1.0 ( Frickey & Lupas, 2004) with a Z-score of 4.5 used as the cut-off threshold.” The Results of that protocol are now reported as follows “The presence of re-entrant loops and the high density of conserved residues within them caused us to examine experimentally characterised re-entrant loops in the PDBTM database. A total of 193 non-redundant re-entrant helices were identified (see Methods). All 193 were clustered with the putative re-entrant loops from Mt2055, Tmem41b and YqjA using relative z-scores derived from an all-against-all DALI run and subsequently clustered in CLANS ( Frickey & Lupas, 2004) with a z-score cut-off of 4.5. The as expected all six re-entrant structures from the query models clustered together. The CLC transporter re-entrant structures of 3orgA (re-entrant 1 and re-entrant 2), 7bxu and 5tqq also clustered with the queries. Additionally, the re-entrant structure from an Undecaprenyl pyrophosphate phosphatase (UppP) (6cb2) also clustered with the queries. UppP is an integral membrane protein that recycles lipid and has structural similarities to CLC transporters (Workman, Worrall, & Strynadka, 2018). Contact maps derived from the pdb files of CLC and UppP structures show the contact map signature corresponding to the re-entrant/TM helix structural feature. Interestingly, the UppP is more similar to the query proteins being only 271 residues in length and having only 6 TM helices.” A list of the 125 chains from which the re-entrant structures were extracted from will be made available in a repository. • Authors state: “The presence of a re-entrant loop packed against each TM helix can also be seen on predicted contact maps for these proteins (Figure 3b).” Re-entrant loops cannot be seen on contact map, only parallel and anti-parallel structures. A similar contact map can be easily generated from 3 transmembrane helices (1 parallel pair and two anti-parallel ones). Yes, a similar contact map feature can be easily generated from 3 transmembrane helices, however, this would result in a box feature of around 20x20 residues (and obviously reflected in the diagonal). Since the re-entrant loop is making contact with itself this can only result in an approximately 10 residue antiparallel feature on the contact map. Only approximately half of the TM helix that is packed with the re-entrant helix will be making contact with the re-entrant loop, therefore, this would result in an additional 10 residue antiparallel feature in addition to a 10-residue parallel feature. Together with the diagonal these will display an approximately 10x10 box feature (also reflected in the diagonal) on the contact map rather than the 20x20 box feature that three transmembrane helices (1 parallel pair and two anti-parallel ones) would produce. This can be seen below; Minor: • Abstract/Results: “The results from the structural bioinformatics analysis of Tmem41b and its homologues showed that they contain a tandem repeat that is clearly visible in evolutionary covariance data but much less so by sequence analysis.” - As I showed above, this statement might not be true. Moreover evolutionary covariance data is the results of sequence analysis, so this sentence is void of sense. Please rephrase. We do not agree with this statement as sequence comparisons and co-variance comparisons are alternative methods to identify tandem repeats. Yes, co-variance is derived from sequence analysis: however, co-variance data contains information that may not be present in data acquired from conventional sequence analysis. • Introduction: “there are eight E. coli representatives of the DedA family (YqjA, YghB, YabI, Yoh, DedA, YdjX, YdjZ, and YqaA)” - Character D is missing in Yoh. Thank you, corrected. • Introduction: “In the current study, we utilised state of the art methods to make structural predictions for two prominent members of the Pfam family PF09335 (Tmem41b and Yqja) by exploiting data derived from sequence, evolutionary covariance and ab initio modelling.” - The most part of the manuscript deal with the sequence analysis of Mt2055, please rephrase this sentence in order to mirror this fact. Thank you, the introduction has been updated to reflect the emphasis on the PF09665 Pfam domain and its representative Mt2055; ‘In the current study, we first linked the Pfam PF09335 family to the PF06695 family and chose a conveniently small Archaeal sequence and then utilised state of the art methods to make structural predictions for not only the Archaeal sequence but also for two prominent members of the Pfam family PF09335 (Tmem41b and Yqja) by exploiting data derived from sequence, evolutionary covariance and ab initio modelling. We are able to predict that both PF09335 homologues (VTT proteins) and PF06995 homologues contain re-entrant loops (stretches of protein that enter the bilayer but exit on the same side of the membrane) as well as a pseudo-inverted repeat topology. The predicted presence of both of these structural features strongly suggests that VTT proteins are secondary active transporters for an uncharacterised substrate.’ • \"Interestingly, each of the re-entrant helices is predicted as a single transmembrane region in the TopCons predictions (see the diagonal of Figure 3b) with a two-residue region of coil in the centre.\" - TOPCONS does not predict coils and such details cannot be seen on the figure - please clarify this sentence. It was not our intention to suggest that Topcons predicts secondary structure. We have changed the paragraph in question clarifying our intention; \"Interestingly, each of the re-entrant helices is predicted as a single transmembrane region in the TopCons predictions. When cross-referenced with the PSIPRED secondary structure prediction it is noted that there is a predicted two-residue region of coil region of coil around the mid-point of the first TM helix prediction. A similar observation can be made for the fourth TM helix prediction with the equivalent coil region being six residues in length (see the diagonal of Figure 4b)\" • The authors should provide the generated PDB files as Supplementary Material. Thank you for pointing out this important omission. Since this journal does not allow Supplementary Material we have deposited the models in a repository now mentioned in the paper. https://figshare.com/articles/dataset/repository_zip/14055212 • Contact map on Figure 7a left is the same that on right (numbering, dots). They should be different if one based on prediction and the other based on experimental data. The left image was generated using predictions and the right with pdb file. They are very similar, but this is to be expected. ClC transporters are a large family and therefore the co-variance-derived predictions will be very accurate." } ] }, { "id": "75805", "date": "20 Jan 2021", "name": "Claudio Bassot", "expertise": [ "Reviewer Expertise Modelling of transmembrane proteins." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors model ab-initio Tmem41b and homologues, characterizing them as secondary transporters. The models are reliable and the study is scientifically robust and worthy of indexing. However, two points are unclear in the text and need to be clarified, plus, some minor changes will improve the readability of the manuscript. Finally, the models could be made available to ensure full reproducibility.\nMajor:\nThe sentence: “The analysis was performed by HELIQUEST (Gautier et al., 2008) which constructed helical wheel diagrams and provided a quantitative measure of the hydrophobic moment for the region being analysed (Figure 4).” is out of context. In that paragraph are described the reentrant helices, shouldn’t the sentence (and the figure) be in the paragraph before where are mentioned the amphipathic helices? The figure discussion in the text should be extended.\n\nThe role of Figure 7 is not clear. It is mentioned in the context of the description of the putative active residues but these are not shown in the figure. Moreover, the only reference to structure 3orgA (shown in the figure) is in the previous paragraph but it’s not related to Figure7. The authors should describe Figure 7 better.\nMinor Introduction:\n“but they only have a 2% representation in the Protein Data Bank (PDB) (Koehler Leman et al., 2015)\". The number of transmembrane proteins has grown significantly in the past few years. From the statistics of PDB and PDBTM the ratio of membrane proteins appears close to 4% now.\nResults:\nFigure 1b could be clearer with the residues numbering on the sequences.\n\nFigure 5. The colours are misleading because they are the opposite of the standard consurf colouration (blue not conserved, purple conserved). The standard colouration would allow a faster understanding of the figure.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6432", "date": "25 Mar 2021", "name": "Daniel Rigden", "role": "Author Response", "response": "The authors model ab-initio Tmem41b and homologues, characterizing them as secondary transporters. The models are reliable and the study is scientifically robust and worthy of indexing. However, two points are unclear in the text and need to be clarified, plus, some minor changes will improve the readability of the manuscript. Finally, the models could be made available to ensure full reproducibility. Models are now available as now mentioned in the text; https://figshare.com/articles/dataset/repository_zip/14055212 Major: The sentence: “The analysis was performed by HELIQUEST (Gautier et al., 2008) which constructed helical wheel diagrams and provided a quantitative measure of the hydrophobic moment for the region being analysed (Figure 4).” is out of context. In that paragraph are described the reentrant helices, shouldn’t the sentence (and the figure) be in the paragraph before where are mentioned the amphipathic helices? The figure discussion in the text should be extended. Yes, we are in agreement with you, the paragraph did seem out of place as well as unfinished. In response we have re-worked the paragraph and noted the helices as merely membrane-parallel at first mention, deferring the question of amphipathicity. “The models ( Figure 3) contained interesting features: two inversely symmetrical repeated units each possessing an amphipathic helix lying parallel to the membrane surface (green) and a re-entrant loop (orange) packed with a TM helix (red).” ….. “In order to test whether the membrane-parallel helices (green in Figure 3) were amphipathic, an analysis of helical wheel diagrams for the fifteen residues preceding the putative re-entrant loops was performed by with HELIQUEST ( Gautier et al., 2008). The quantitative measures of the hydrophobic moment for the regions being analysed ( Figure 5) support that they are indeed amphipathic helices. The hydrophobic moments ranged from 0.298 to 0.546.” The role of Figure 7 is not clear. It is mentioned in the context of the description of the putative active residues but these are not shown in the figure. Moreover, the only reference to structure 3orgA (shown in the figure) is in the previous paragraph but it’s not related to Figure7. The authors should describe Figure 7 better. Thank you for pointing out the ambiguity of Figure 7. There was an error in the figure numbering resulting in Figure 7 not being cited in the main text. This has now been amended and the relevance of figure 7 is highlighted in the following paragraph; “Analysis of the Cl -/H + antiporter structures show that they contain a similar inverted repeat as we infer for the DedA homologues, resulting in pseudo-2-fold axis of symmetry running along the membrane ( Duran & Meiler, 2013). Again similarly, the Cl -/H + antiporter 3orgA also contains the amphipathic helices on the N-terminal side of the re-entrant loops. The fact that the presence of the amphipathic helices is restricted only to 3orgA and not found in all homologues suggest that these features are not essential for function (Figure 7). “ Minor Introduction: “but they only have a 2% representation in the Protein Data Bank (PDB) (Koehler Leman et al., 2015)\". The number of transmembrane proteins has grown significantly in the past few years. From the statistics of PDB and PDBTM the ratio of membrane proteins appears close to 4% now. As suggested using the PDB & PDBTM stats we can see the ratio of membrane proteins is above the 2% previously quoted. The PDBTM has 5785 entries with a total of 174507 entries for the PDB. I have updated the introduction accordingly; “For instance, membrane proteins are encoded by 30% of the protein-coding genes of the human genome ( Almén et al., 2009), but they only have a 3.3% representation in the Protein Data Bank (PDB) (5785 membrane proteins out of 174507 PDB entries).” Results: Figure 1b could be clearer with the residues numbering on the sequences. Yes agreed. Numbering has been added to figure 1b (now 2b, due to the inclusion of an additional figure in the revised manuscript). It can be seen that the additional detail of the numbering makes it easier to cross reference the images that make up this figure. The new figure is shown; Figure 5. The colours are misleading because they are the opposite of the standard consurf colouration (blue not conserved, purple conserved). The standard colouration would allow a faster understanding of the figure. We agree. Colouring on the B-factor column directly produces the results we show but the new blue-purple spectrum does seem to be well-adopted. We have therefore replaced the figure and updated the legend to read “trRosetta models with Consurf conservation mapping for (a) Mt2055 (b) Tmem41b (c) Yqja. Conservation is shown as a spectrum from purple (highly conserved) to blue (not conserved).”" } ] } ]	1	https://f1000research.com/articles/9-1395
https://f1000research.com/articles/7-230/v1	27 Feb 18	{ "type": "Research Article", "title": "Biochemical and clinical characterization of metabolic phenotypes: a cross-sectional study from Maracaibo city, Venezuela", "authors": [ "Valmore Bermudez", "Joselyn Rojas", "Juan Salazar", "Maria Sofia Martinez", "Luis Carlos Olivar", "Maria Jose Calvo", "Andres Mindiola", "Roberto Añez", "Sandra Wilches-Duran", "Marcos Cerda", "Modesto Graterol", "Rosemily Graterol", "Juan Diego Hernandez", "Carlos Garicano", "Manuel Velasco", "Joselyn Rojas", "Maria Sofia Martinez", "Luis Carlos Olivar", "Maria Jose Calvo", "Andres Mindiola", "Roberto Añez", "Sandra Wilches-Duran", "Marcos Cerda", "Modesto Graterol", "Rosemily Graterol", "Juan Diego Hernandez", "Carlos Garicano", "Manuel Velasco" ], "abstract": "Background: In 1980, Reuben Andresen observed that in certain individuals, obesity did not increase mortality, introducing an atypical phenotype called “healthy obese”. Other studies reported that 10-15 % of lean individuals presented insulin resistance, hyperglycemia and dyslipidemia. The objective of this study was to evaluate biochemical and clinical characteristics of metabolic phenotypes in Maracaibo city. Methods: A descriptive, cross-sectional study with a randomized multistage sampling was performed including 1226 non diabetic individuals from both sexes. For phenotype definition, the subjects were first classified according to their BMI into Normal-Weight, Overweight and Obese; then divided in metabolically healthy and unhealthy using a two-step analysis cluster. To evaluate the relationship with coronary risk, a multiple logistic regression model was performed. Results: In the studied population, 5.2% (n=64) corresponded to unhealthy lean subjects, and 17.4% (n=217) to healthy obese subjects. Metabolically unhealthy normal-weight (MUNW) phenotype was found in males in 53.3% in contrast to 51.3% of metabolically unhealthy obese (MUO) phenotype found in females. An association between metabolically unhealthy phenotypes and a higher risk of a coronary event was found, especially for obese individuals (MHO: OR=1.85 CI95%: 1.11-3.09; p=0.02 and MUO: OR=2.09 CI95%: 1.34-3.28; p<0.01). Conclusion: Individuals with atypical metabolic phenotypes exist in Maracaibo city. Related factors may include insulin resistance, basal glucose levels, and triglycerides levels. Lastly, cardiovascular risk exhibited by healthy obese individuals should be classified in categories of major coronary risk related to lean subjects.", "keywords": [ "Metabolic phenotypes", "two-step cluster", "metabolically unhealthy lean", "metabolically healthy obese", "coronary risk" ], "content": "Introduction\n\nObesity is considered an entity with major morbi-mortality in the world since the end of the 20th century1. Multiples studies have shown its role as an independent risk factor for various cardiometabolic disorders such as hypertension (HTN), dyslipidemias, Type 2 Diabetes Mellitus (T2DM) and cardiovascular disease (CVD)2. For this reason, the actual clinical practice catalogues an obese patient as an “unhealthy” patient and a lean patient is considered “healthy”.\n\nIn spite of this, in 1980, Reuben Andresen discovered that in certain groups of individuals the obesity was not a mortality increasing factor, introducing the subtype “Healthy Obese”3. Around 20 years later, Ferranini et al. observed that a group of certain obese nondiabetic non-hypertensive subjects presented low insulin resistance (IR) prevalence, suggesting that this subtype must have a different risk of having T2DM and CVD from the IR obese; also suggesting a different management for them4.\n\nFurthermore, in 1975, Bernstein et al. observed that 11 normal-weight men with type IV or V dyslipidemia presented higher serum glucose levels; and also carried bigger sized adipocytes with respect to their healthy counterparts5. Years later, Ruderman et al. introduced the “Metabolically Unhealthy Normal-Weight” phenotype attributed to lean individuals with metabolic alterations associated to obesity6.\n\nThe importance of these atypical metabolic phenotypes lies in the fact that their diagnosis may be challenging for clinicians delaying their detection. Because of this, in recent years, multiple studies have been dedicated to the research of accurate clinical, biochemical, and genetic elements capable to detect these atypical metabolic states, and their evolution.\n\nIn this sense, these phenotypes determinants and frequencies have not been deeply researched in Latin-American populations7. The objective of this study is to characterize, from a clinical-biological point of view, the metabolic phenotypes in the population from Maracaibo city, Venezuela.\n\n\nMaterials and methods\n\nThe Maracaibo City Metabolic Syndrome Prevalence Study (MMSPS) is a cross-sectional study whose purpose is to detect metabolic syndrome and cardiovascular disease risk factors in the adult population from Maracaibo, the second largest city of Venezuela, with approximately 2,500,000 inhabitants, during the period May 2007 – December 2009. The original study included a total of 2230 individuals of both genders, aged between 18–85 years old, and the study protocol was previously reported8. This sub-analysis excluded those individuals with no measurements of serum insulin levels. Patients with past history of diabetes were also excluded because their disease control, evolution and pharmacological treatments would affect the variables in the study.\n\nThese subjects were categorized into six groups, first according to their Body Mass Index (BMI) (normal-weight, overweight and obese) and second, to their healthy/unhealthy definition. This categorization was made using the protocol from two-step cluster analysis published previously9. The metabolic variables were chosen as possible metabolic predictors based on their physiological function and biological plausibility. These variables were: mean arterial pressure (MAP), triglycerides (TAG), total cholesterol, HDL-C, HOMA2-IR, HOMA2-βcell, HOMA2-S, fasting blood glucose, non-HDL-C cholesterol, TAG/HDL-C ratio, and high-sensitivity C-Reactive Protein (hs-CRP) levels; waist circumference (WC) was excluded and was assessed as a dependent variable. The predictive strength of these variables was analyzed in accordance to cluster ability and quality, ranging from 0.0 to 1.0. The most appropriate predictive variables selected for each group were: (a) HOMA2-IR and HOMA2-βcell for normal-weight women; (b) HOMA2-IR, HOMA2-βcell and TAG for normal-weight men; (c) HOMA2-IR and HOMA2-βcell for overweight women; (d) HOMA2-IR, HOMA2-βcell, and TAG for overweight men; and (e) HOMA2-IR for male and female obese patients. The two-step cluster analysis with SPSS was conducted in two phases: during the first step (called “precluster”), the subjects were divided into several small subclusters. Then, the obtained subclusters were grouped in preferred number of clusters; if the desired number of clusters was unknown, the SPSS two-step cluster component would find the proper number of clusters automatically. Once the program analyzed the subclusters with the characteristics of each BMI category (as described previously), the subjects were categorized into 6 phenotypes: healthy normal-weight (HNW), metabolically unhealthy normal-weight (MUNW), healthy and metabolically disturbed overweight, metabolically unhealthy obese (MUO), and metabolically healthy obese (MHO). Overweight subjects were excluded from this secondary analysis since they represent a non-conventional group outside the metabolic phenotypes and require separate analysis. The final sample included 1226 subjects (Figure 1).\n\nDuring simple selection, subjects with no measurements of serum insulin levels and patients with past history of diabetes were excluded. These subjects were categorized into six groups, first according to their BMI and second to their healthy/unhealthy definition, using two-step cluster analysis.\n\nData was collected through completion of a full clinical record carried out by trained personnel, which included interrogation regarding ethnic origin and socioeconomic status by the Graffar scale according to Méndez-Castellano10. The assessment of blood pressure was done by applying the auscultatory technique, and HTN classification was made using the criteria proposed in the VII Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure11.\n\nFor Anthropometric Analysis, an electrical bioelectric scale was used to obtain weight (Tanita, TBF-310 GS Body Composition Analyzer, Tokyo – Japan). Height was measured using a calibrated metric measurement tape, with the subject standing up barefoot. BMI formula (weight/height2) was applied, expressing the results as kg/m2. Obesity was classified applying the WHO criteria12 based on the BMI value. Finally, WC was measured using calibrated measuring tape in accordance to the anatomical landmarks proposed by the USA National Institutes of Health protocol13.\n\nPhysical activity. Physical activity (PA) was assessed with the International Physical Activity Questionnaire (IPAQ). For statistical analysis, PA was evaluated in 4 domains: occupational, household, transport, and leisure. In each of these domains, subjects were categorized as follows: (a) inactive, MET/week = 0, or (b) active, MET/week > 0. The latter were then subcategorized by gender-specific MET/week quintiles in each domain (Table 1), which were published previously14.\n\nMaracaibo city, Venezuela.\n\nObtained from IPAQ scoring. Subjects with 0 MET were excluded from quintiles and classified separately as Inactive.\n\nFasting levels of glucose, cholesterol, TAG, HDL-C, and hs-CRP were assessed in our clinical laboratory using an automatized computer analyzer (Human Gesellschaft fur Biochemica und Diagnostica mbH). LDL-C and VLDL-C levels were calculated applying the Friedewald formulas15. When TAG were over 400 mg/dL measurement was done using lipoprotein electrophoresis and optical densitometry (BioRad GS-800 densitometer, USA). Lipoprotein (a) [Lp(a)] was estimated through the latex turbidimetric method, Human Gesellschaft für Biochemica and Diagnostica, Germany. Likewise, serum hs-CRP levels were quantified employing immunoturbidimetric essays (Human Gesellschaft für Biochemica and Diagnostica MBH). Insulin was determined using an ultrasensitive ELISA method (DRG Instruments GmbH, Germany, International DRG Division, Inc.). For the evaluation of insulin resistance (IR), the HOMA2-IR model proposed by Levy et al. was utilized16 determined through the HOMA-Calculator v2.2.2 program. Visceral Adiposity Index (VAI) calculation was performed with the gender-specific equations proposed by Amato et al.17. The Metabolic Syndrome (MS) diagnosis was done using the Harmonizing-2009 consensus criteria18.\n\nFor proper equation calibration, the constants in the formula regarding major cumulative coronary events (lethal and non-lethal myocardial infarction, symptomatic and no symptomatic angina) were substituted with the local statistics obtained from the Vital Statistics Yearbook of the State of Zulia from 2008, where the morbidity and mortality for cardiovascular diseases is registered, the calibration process has been detailed previously19. The coronary risk was classified in 2 categories: <5% in 10 years, and ≥5% in 10 years.\n\nNormal distribution of continuous variables was assessed using Geary’s test; for normally distributed variables, the results were expressed as arithmetic mean ± SD (standard deviation). Variables without normal distribution were logarithmically transformed, and normal distribution subsequently corroborated. When normalization could not be achieved, these variables were expressed as medians (25th percentile–75th percentile). Student’s –test/One-way ANOVA or Mann-Whitney/Kruskal Wallis’s tests were applied to evaluate differences between means or medians, respectively. Qualitative variables were expressed as absolute and relative frequencies, assessed through the χ2 test and the Z test for Proportions.\n\nA logistic regression model was constructed with coronary risk as dependent variable and independent variables: gender, age groups, ethnicity, socioeconomic status, smoking habit, physical activity in leisure time, elevated TAG, and metabolic phenotypes. Database construction and statistical analysis were done using the Statistical Package for the Social Sciences (SPSS) v22 for Windows (IBM Inc., Chicago, IL), results were considered statistically significant when p<0.05.\n\n\nResults\n\nA total of 1226 individuals were studied, 55.1% (n=676) corresponded to females and 44.9% (n=550) to males. The mean age (years) of the general population was 37.94±14.99. Subjects distribution according to their metabolic phenotype is shown in Figure 2 where the 5.2% (n=64) of the individuals were classified as MUNW, and 17.4% (n=213) as MHO, representing 34.13% from the total of obese subjects, while sociodemographic and metabolic characteristics from the studied simple are shown in Table 2.\n\nFor this sub-analysis overweight subjects were excluded, evaluating only the typical obesity phenotypes with 4 groups.\n\nMaracaibo city, Venezuela.\n\n‡ Past history and Diagnosed in the Study\n\n Metabolic Syndrome Diagnosis according to 2009 Harmonizing Consensus\n\n† HOMA2-IR ≥2\n\nIn the evaluation of the epidemiologic behavior of the metabolic phenotypes according to sex, we found that HNW and MUO individuals were predominately females (62.5%, n=336; 51.3%, n=211 respectively), while the atypical phenotypes were predominately males (MUNW: 56.3%, n=36; MHO: 52.6%, n=112. χ2=22.53, p<0.001). Likewise, a statistically significant association was found between age groups and metabolic phenotypes (χ2= 211.91, p<0.001), observing a predominance in the < 30 years age group in the normal-weight phenotype (HNW: 56.1%, n=302; MUNW: 57.8%, n=37), whereas the 30–49 age group was predominately obese phenotypes (MHO: 47.9%, n=102; MUO: 50.1%, n=106). There was no statistically significant association between metabolic phenotypes, ethnic groups (χ2= 20.96, p=0.05) and socioeconomic status (χ2= 14.56, p=0.27) (Table 3).\n\nMaracaibo city, Venezuela.\n\nHNW (Healthy Normal Weight); MUNW (Metabolically Unhealthy Normal Weight); MHO (Metabolically Healthy Obese); MUO (Metabolically Unhealthy Obese).\n\n* Chi-Square Test.\n\n** Z-test of proportions.\n\nInitially, in relation to the smoking habit, the non-smokers were the most frequent group (χ2=30.91; p<0.001), despite the fact MUNW phenotype consisted of the highest percentage of smoking individuals (18.8%, n=12), whereas MUO subjects consisted of the highest proportion of past smoking subjects (20.2%, n=83). On the other side, in the evaluation of the metabolic phenotypes according to PA there was a statistically significant association in the transport-related physical activity (χ2=43.39; p<0.001) and leisure activities (χ2=50.48; p<0.001) (Table 4).\n\nMaracaibo city, Venezuela.\n\nHNW (Healthy Normal Weight); MUNW (Metabolically Unhealthy Normal Weight); MHO (Metabolically Healthy Obese); MUO (Metabolically Unhealthy Obese).\n\n* Chi-Square Test.\n\n** Z-test of proportions.\n\nDistribution of subjects according to phenotypes and endocrine-metabolic alterations are shown in Table 5. A high percentage of MUNW and MUO individuals with insulin resistance was found in contrast to healthy subjects (79.7%, n=51 and 97.1%, n=399, respectively). On the other side, a higher percentage of MUNW with high TAG was found (34.4% n=22 vs 9.5% n=51 HNW; p<0.05) and also a higher prevalence of MS (29.7% n=19 vs 12.3% n=66; p<0.05 HNW); similar findings were observed in the obese phenotypes, where a minor prevalence of these alterations were found in the MHO subjects (high TAG levels: 28.8% n=60 vs 42.8% n=176, p<0.05; MS: 53.1% n=113 vs 69.3% n=285, p<0.05). Finally, a significant association was found between the metabolic phenotypes with low HDL-C (χ2=44.08; p<0.0001) and HTN (χ2= 182.22, p<0.0001).\n\nHNW (Healthy Normal Weight); MUNW (Metabolically Unhealthy Normal Weight); MHO (Metabolically Healthy Obese); MUO (Metabolically Unhealthy Obese).\n\n* Chi-Square Test.\n\n** Z-test of proportions.\n\n‡Personal history and Diagnosis in the Study\n\nBiochemical and clinical characteristics according to metabolic phenotypes are shown in Table 6. An increasing tendency of their variable levels was observed, except on HOMA2-IR, HOMA2-βcell, HOMA2-S, insulin y glucose levels.\n\nMaracaibo city, Venezuela.\n\nHNW (Healthy Normal Weight); MUNW (Metabolically Unhealthy Normal Weight); MHO (Metabolically Healthy Obese); MUO (Metabolically Unhealthy Obese).\n\nSD=Standar Deviation;\n\n* One-way ANOVA Test\n\n¶ As Median (p25–p75th) Comparison: Kruskal Wallis Test\n\nAn association between metabolically unhealthy phenotypes and a higher risk of a coronary event was found. This association was stronger for unhealthy phenotypes than for their healthy counterparts. However, results were statistically significant for obese individuals (MHO: OR=1.85 CI95%: 1.11-3.09; p=0.02 and MUO: OR=2.09 CI95%: 1.34-3.28; p<0.01) (Table 7).\n\nMaracaibo city, Venezuela.\n\na Confidence Interval (95%); b Level of significance\n\nDependent Variable: Coronary risk: <5% in 10 years vs ≥5% in 10 years\n\n* Adjusted Model for: sex, age, ethnic group, socioeconomic status, smoking habit, physical activity in leisure dimension according to IPAQ, high TAG, and metabolic phenotypes.\n\n\nDiscussion\n\nObesity is a prioritized area for the world health systems because of its increasing prevalence, incidence, and associated costs in the last decade20. This disease has been defined classically as “excessive presence of adipose tissue that is injurious for health” and given its association to other chronic-degenerative diseases3,21 has been stereotyped as “more adiposity, more risk”. All the classic methods employed for obesity diagnosis, even central and global, are indirect measurements. For different populations they do not allow to determine the adipose tissue functioning from individuals, even though they have high sensitivity, specificity, and predictive values. Based on this, multiple epidemiologic studies have detected a considerable percentage of individuals who did not enter in the classic “HNW” and “MUO” phenotypes, showing the existence of atypical metabolic phenotypes called “MUNW” and “MHO”3. The defining criteria of these metabolic states differ significantly between studies and are defined under highly subjectivity levels, nonetheless insulin sensitivity and lipid profile are often used to define healthy and unhealthy phenotypes22–24.\n\nGiving this criteria and methods discrepancy, such as the psychobiologic, sociodemographic, and genetic patterns according to latitudes, the phenotype frequency presents high variability25. This could bias the study by selecting predetermined variables and cut-off points to consider an individual as healthy or unhealthy. In this sense, data mining techniques were proposed to avoid potential bias. The program would group subjects according to spontaneous tendencies and biologic behavior of related variables.\n\nApplied studies in Asia reported a prevalence of 8.7%–13.07% and 3.9%–15.5% for MUNW and MHO phenotypes, respectively26,27. Likewise, studies conducted in Europe reported frequencies ranging between 18.9% and 45.8% for the MUNW phenotype, and between 2.1% and 18.5% for the MHO phenotype28–30; a similar variability was observed in American research studies31,32. Latin American reports are scant, however Fanghanel et al.33 showed a 5.8% prevalence of the MUNW phenotype for the Mexico City, similar to the one showed in the present study, whereas contrasting the obese phenotypes the Maracaibo population exhibited the highest prevalence of MHO subjects (17% vs 10.8% of the Mexican population).\n\nThe atypical metabolic phenotypes, as MUNW and MHO, tend to be observed in females with more frequency32,34. However, the present study reported these phenotypes were more frequent in males. Significant difference between sexes was found in the MUNW group, similar to the study by Hinnouko et al.35. Smoking habit, age, and physical activity values, were discovered as influencing factors in these findings.\n\nIn the same manner, multiple studies have reported that healthy phenotype prevalence decreases with age27,29, but in our population an increase was observed in the frequency of MHO individuals older than 30 years old. Yoo et al.36 did not report differences in this phenotype prevalence between subjects older and younger than 35 years. Regarding the MUNW phenotype in the Maracaibo population, a higher frequency was found in subjects younger than 30 years. A considerable part of epidemiologic studies that evaluate this association possessed samples conformed by subjects older than 35 years. This may limit the establishment of a tendency in frequency of healthy phenotypes according to age. Similarly, factors such as ethnicity from African descendants37 and socioeconomical status38 have been related to the presence of atypical phenotypes, but no relationship was found between these variables in Maracaibo population.\n\nOne of the greatest enigmas formulated in relation to the atypical metabolic phenotypes, is focused on its conditioning factors. Psychobiologic habits have been considered key elements in comprehension of its biology and behavior related to time. Diniz et al.39 found a significant association between healthy metabolic phenotypes with absence of smoking habit, also with increased PA levels, such as the present study. Ortega et al.40 reported that MHO subjects present with better cardiorespiratory fitness profiles than their unhealthy counterpart, and by adjusting for this variable the MHO individuals showed less mortality. Other studies report that the phenotypes progression from health to unhealthiness is not related to the smoking habit, alcohol, or quantified PA through indirect methods30 and depends fundamentally on abdominal circumference and visceral adiposity increment.\n\nRegarding to cardiometabolic profiles, our study showed evidence of significantly higher HOMA2-βcell values in all of the unhealthy phenotypes, described previously by the NHANES study41 and by Madeira et al.42. Also higher HOMA2-IR and a lower HOMA2-S demonstrate again the importance to define metabolic states in lean and obese individuals. They could also elevate the risk of developing T2DM and CVD in the unhealthy phenotypes, given their hyper functioning pancreatic beta cell and hyperinsulinemia43.\n\nMHO subjects present with lower HOMA2-IR and higher TAG, LDL-C, PAS, PAD, and hs-CRP levels. In contrast to lean subjects, MHO has higher VAI. The latter constitutes an initial obesity state, without a significant risk of T2DM and CVD in the short term (7–11 years)44, but there is in the long term (>16–30 years)45. The natural history of the MHO is variable, only 16% of MHO individuals stay on that status without alteration for the following 7–8 years46. Those who progress to an unhealthy state present a higher risk of high blood pressure, low-grade inflammation, bad metabolic control and high TAG30. In spite of the metabolic “benign” state of the MHO adipose tissue, non-metabolic complications of obesity, do not exclude these subjects from getting T2DM, CVD, and chronic diseases associated with obesity in the future34,35.\n\nHealthy obese individuals must be classified in categories with higher risk of a coronary event compared to lean subjects. This is consistent with previous reports related to metabolic phenotypes and CVD, suggesting that healthy obese subjects have a higher risk profile in comparison to those with lower BMI36; as well as an increased risk for CVD47 and metabolic disorders such as fatty liver and low-grade inflammation48. Given the above, a profound evaluation of these patients is recommended. This includes not only obese subjects but also those who are overweight, which can go unnoticed in a routine consultation and CVD could be subclinical; as it has been demonstrated by Khan et al. in 475 women from the SWAN study49.\n\nFinally, despite the fact that our report presents a novel method to classify healthy and unhealthy subjects, it is important to mention the difficulty to follow-up these individuals. The latter would show the atypical phenotype stability related to time, as well as the incidence of T2DM and CVD. This was the main limitation of our study. In addition our study lacks nutritional data. For this reason, a thorough and constant evaluation of subjects with atypical metabolic phenotypes is recommended, given their demonstrated unsteadiness in time, and associated non metabolic comorbidities observed especially in the MHO individuals.\n\n\nData availability\n\nDataset 1: MMSPS metabolic phenotype dataset. BMI: Body Mass Index, WaistC: Waist Circumference, HDL-C: High Density Lipoprotein Cholesterol, VLDL-C: Very Low Density Lipoprotein Cholesterol, LDL-C: Low Density Lipoprotein Cholesterol, Lp(a): Lipoprotein (a), hs-CRP: high Sensitivity C Reactive Protein, Non-HDL-Col: Non-High Density Lipoprotein Cholesterol, TAG/HDL ratio: Triglycerides/High Density Lipoprotein ratio VAI: Visceral Adiposity Index, BP: Blood Pressure, HNW: Healthy Normal-Weight, MUNW: Metabolically Unhealthy Normal-Weight, MUO: Metabolically Unhealthy Obese, MHO: Metabolically Healthy Obese. 10.5256/f1000research.13897.d19335150\n\n\nEthics and consent\n\nThe study was approved by the Bioethics Committee of the Endocrine and Metabolic Research Center – University of Zulia (approval number: BEC-006-0305). This ethical approval included all future studies that used the data from the Maracaibo City Metabolic Syndrome Prevalence Study (MMSPS). All participants signed written a informed consent for participation in the study before being questioned and physically examined by a trained team.\n\n\nAbbreviations\n\nCVD: cardiovascular disease\n\nHDL-C: High Density Lipoprotein - Cholesterol\n\nHNW: healthy normal-weight\n\nHOMA: Homeostasis Model Assesment\n\nHTN: hypertension\n\nhs-CRP: high-sensitivity C-Reactive Protein\n\nIR: insulin resistance\n\nLDL-C: Low Density Lipoprotein – Cholesterol\n\nMAP: mean arterial pressure\n\nMET: Metabolic Equivalent\n\nMMSPS: Maracaibo City Metabolic Syndrome Prevalence Study\n\nMHO: metabolically healthy obese\n\nMS: Metabolic Syndrome\n\nMUNW: metabolically unhealthy normal-weight\n\nMUO: metabolically unhealthy obese\n\nPA: Physical activity\n\nSD: standard deviation\n\nTAG: triglycerides\n\nT2DM: Type 2 Diabetes Mellitus\n\nVAI: Visceral Adiposity Index", "appendix": "Competing interests\n\n\n\nThe authors have no conflicts of interest to disclose.\n\n\nGrant information\n\nThis work was supported by research grant Nº CC-0437-10-21-09-10 from the Technological, Humanistic, and Scientific Development Council (Consejo de Desarrollo Científico, Humanístico y Tecnológico; CONDES), University of Zulia.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nWorld Health Organization (WHO): Obesity and overweight: Fact Sheet. 2016; [cited 2017 Mar 20]. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYoo HK, Choi EY, Park EW, et al.: Comparison of Metabolic Characteristics of Metabolically Healthy but Obese (MHO) Middle-Aged Men According to Different Criteria. Korean J Fam Med. 2013; 34(1): 19–26. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchmiegelow MD, Hedlin H, Mackey RH, et al.: Race and ethnicity, obesity, metabolic health, and risk of cardiovascular disease in postmenopausal women. J Am Heart Assoc. 2015; 4(5): pii:e001695. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYang HK, Han K, Kwon HS, et al.: Obesity, metabolic health, and mortality in adults: a nationwide population-based study in Korea. Sci Rep. 2016; 6(6): 30329. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDiniz Mde F, Beleigoli AM, Ribeiro AL, et al.: Factors associated with metabolically healthy status in obesity, overweight, and normal weight at baseline of ELSA-Brasil. Medicine (Baltimore). 2016; 95(27): e4010. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOrtega FB, Lee DC, Katzmarzyk PT, et al.: The intriguing metabolically healthy but obese phenotype: cardiovascular prognosis and role of fitness. Eur Heart J. 2013; 34(5): 389–97. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRomero-Corral A, Somers VK, Sierra-Johnson J, et al.: Normal weight obesity: a risk factor for cardiometabolic dysregulation and cardiovascular mortality. Eur Heart J. 2010; 31(6): 737–46. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMadeira FB, Silva AA, Veloso HF, et al.: Normal weight obesity is associated with metabolic syndrome and insulin resistance in young adults from a middle-income country. PLoS One. 2013; 8(3): e60673. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSuccurro E, Marini MA, Frontoni S, et al.: Insulin secretion in metabolically obese, but normal weight, and in metabolically healthy but obese individuals. Obes (Silver Spring). 2008; 16(8): 1881–6. PubMed Abstract \| Publisher Full Text\n\nOgorodnikova AD, Kim M, McGinn AP, et al.: Incident cardiovascular disease events in metabolically benign obese individuals. Obes (Silver Spring). 2012; 20(3): 651–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHansen L, Netterstrøm MK, Johansen NB, et al.: Metabolically Healthy Obesity and Ischemic Heart Disease: A 10-Year Follow-Up of the Inter99 Study. J Clin Endocrinol Metab. 2017; 102(6): 1934–1942. PubMed Abstract \| Publisher Full Text\n\nKim NH, Seo JA, Cho H, et al.: Risk of the Development of Diabetes and Cardiovascular Disease in Metabolically Healthy Obese People: The Korean Genome and Epidemiology Study. Medicine (Baltimore). 2016; 95(15): e3384. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDurward CM, Hartman TJ, Nickols-Richardson SM: All-cause mortality risk of metabolically healthy obese individuals in NHANES III. J Obes. 2012; 2012: 460321. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShaharyar S, Roberson LL, Jamal O, et al.: Obesity and metabolic phenotypes (metabolically healthy and unhealthy variants) are significantly associated with prevalence of elevated C-reactive protein and hepatic steatosis in a large healthy Brazilian population. J Obes. 2015; 2015: 178526. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKhan UI, Wang D, Thurston RC, et al.: Burden of subclinical cardiovascular disease in “metabolically benign” and “at-risk” overweight and obese women: the Study of Women’s Health Across the Nation (SWAN). Atherosclerosis. 2011; 217(1): 179–86. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBermudez V, Rojas J, Salazar J, et al.: Dataset 1 in: Biochemical and clinical characterization of metabolic phenotypes: a cross-sectional study from Maracaibo city, Venezuela. F1000Research. 2018. Data Source" }	[ { "id": "35881", "date": "19 Jul 2018", "name": "Víctor A. Cortes", "expertise": [ "Reviewer Expertise Basic and clinical research on adipose tissue disorders", "diabetes", "dyslipidemia and fatty liver" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors aimed to estimate the prevalence of “atypical metabolic phenotypes” i.e. lean metabolically unhealthy and obese metabolically healthy individuals in the city area of Maracaibo, Venezuela. For that, they analyzed previously generated data from a cross-sectional city-wide health survey (MMSPS), grouping individuals in three BMI categories (normal weight, overweight and obese) and in two “metabolic health” categories (healthy and unhealthy).\nOverall, this study is incremental, reporting no new scientific information on the metabolic phenotypes whatsoever. Its relevance relay on the fact that is methodologically correct study of a Latin American population, which is largely underrepresented in the international literature.\nNevertheless, there are several specific caveats that must be addressed before the article be suitable for indexing:\n\nAbstract: Methodololgy:\nIt must tell the source of the sample to make clear that is a city-wide health survey. It must indicate what was the specific criteria for this clustering individuals in healthy/unhealthy categories. It is not enough to tell that is was made upon a 2-step clustering analysis.\nResults:\nIt should be re written to clearly indicate the frequency of each phenotype category and indicate if there were differences between the sexes in this distribution and the OR for casrdiovascular risk factors or diseases. It must indicate what is the difference between unhealthy lean and unhealthy normal-weight individuals\nConclusions: There are no reasons to suppose that individuals of Maracaibo city will have no “atypical metabolic phenotypes” as the rest of the world populations, thus the authors should rephrase this sentence to make more scientifically sound. Also, the conclusion relative to the increased cardiovascular risk of “healthy obese” individuals should be better explained since it cannot be derived from the data reported in the Results section of the abstract.\n\nIntroduction: The phrase: “For this reason, the actual clinical practice catalogues an obese patient as an “unhealthy” patient and a lean patient is considered “healthy”, should be modified to make its medical meaning clearer, because it is evident for everybody that many lean people are unhealthy.\n\nAlso, the paper will gain interest if the authors comment what is the importance of researching the “atypical phenotypes”in general. For example, is there any evidence that these individuals can be misclassified in their cardiometabolic risk based solely in the BMI?\n\nFinally, it is important that the authors comment the extent to what Maracaibo city population is representative of other Latin American populations. For international readers will be interesting to learn that American populations are extremely heterogenous in both genetic and cultural aspects.\n\nMaterials and methods: “Population selection”: this whole methodological section is cryptic and is basically a summary of the published in the reference 9. I suggest to re write it to make more understandable for general readers. Specifically, it must be justified why the authors did choose not to use more a conventional definition of metabolic health, such as the metabolic syndrome definition used by ATP III guidelines.\n\n“Physical activity”: it should be improved the explanation of what is the relevance and connection of table 1 with the rest of the paper. Also, in this table there is no quantitative definition of the “Work domain” and “lower/upper limit” categories. Information of the proportion of each category over the overall would be useful to summarize these data.\n“Calibration of the Framingham-Wilson equation and coronary risk categorization for the population of Maracaibo city”: the authors must explain what asymptomatic angina is, since medically angina is a symptom by itself.\n\nResults: Table 2: change “Indian-american” for “native American”, if its corresponds since “Indian” correspond to India nationals\n“Metabolic phenotypes and biologic-anthropometric variables” section: Since all the variables in this table are statistically different, a post test comparing individual groups should be important to make sense of the noted global differences.\n\nAlso, a better description and explanation of these particular results is required in the main text.\n“Metabolic phenotypes and coronary risk classification” section: the meaning of the first phrase must be clarified since only obese individuals showed increased OR: other comparisons were not statistically significant with the adjusted model, thus were not different.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/7-230
https://f1000research.com/articles/10-239/v1	24 Mar 21	{ "type": "Research Article", "title": "Effect of teaching leadership styles on the prevention of adolescence pregnancy: a case study conducted at Universidad César Vallejo, Peru", "authors": [ "José Manuel Delgado Bardales", "Edward Freddy Rubio Luna Victoria", "Orfelina Valera Vega", "Rosa Mabel Contreras Julián", "Gabriela del Pilar Palomino Alvarado", "Keller Sánchez Dávila", "Yoni Meni Rodríguez Espejo", "Daniel A. Lowy", "Edward Freddy Rubio Luna Victoria", "Orfelina Valera Vega", "Gabriela del Pilar Palomino Alvarado", "Keller Sánchez Dávila", "Yoni Meni Rodríguez Espejo", "Daniel A. Lowy" ], "abstract": "Background: In this study we aimed to determine the effect of university teaching and leadership styles at Universidad César Vallejo on the prevention of adolescence pregnancy. Methods: We performed a non-experimental, quantitative, cross-sectional study, with a correlational descriptive design using a survey. Participants were students in classes enrolled in the starting two semesters at Universidad César Vallejo, Peru. The questionnaire comprised three sections: (I) General demographic data, (II) Teacher leadership styles, and (III) Pregnancy prevention teachings. Results: According to the students (n=793), teaching leadership styles are of three types: Laissez faire, transactional, and transformational. Types of pregnancy prevention from the students’ standpoint are the following: \"high\" level of \"primary prevention\" (44.4%), \"medium\" level of \"tertiary prevention\" (36.6%), and \"high\" level of \"secondary prevention\" (36.2%). Chi-Square test revealed that the three styles of leadership significantly affect the prevention of pregnancy: transformational (X2 = 136.390), transactional (X2 = 95.539), and Laissez Faire (X2 = 80.557) (p <0.05). Overall, there is a significant impact of university teaching leadership on pregnancy prevention among higher education students (X2 = 110,634, p = 0.0000), with a significance level of 5%. Conclusions: It can be concluded that for college students, teaching leadership style significantly affects the prevention of adolescent pregnancy.", "keywords": [ "leadership", "university teacher", "pregnancy prevention", "public health" ], "content": "Introduction\n\nIn low- and middle-income countries, there are about 16 million young women, aged 15–19, and approximately 1 million girls less than 15 years old, who give birth over one year. Problems that occur during pregnancy and childbirth are the second leading cause of death of these women, worldwide. Also, every year about 3 million girls aged 15–19 undergo unsafe abortions (UNICEF, 2014). For this reason, pregnancy of adolescents and young women is a significant public health issue in South America, as it reduces the chances of educational development of pregnant woman and by this, it amplifies the risk of their poverty. To address these issues, there is an increasing number of studies that examine the role of teachers and lecturers in sexual education and prevention in the Latin American region (Anzoategui, 2015; Arroyave et al., 2015; Newman et al., 2008; Rosario, 2012). According to Arroyave & co-workers (2015), students are aware of the demanding responsibility of motherhood and fatherhood, nevertheless, they do not practice safe sex. Ramírez & Contreras (2013) emphasized that unfortunately, high school and university professors do not have sufficient knowledge related to this topic, for example, the ways in which HIV is transmitted. Therefore, they are not prepared to provide efficient sexual education. Reátegui & Carranza (2016) concluded that teaching style was likely the most important contributor to preventing adolescent pregnancy. Here, we present a correlation analysis between teaching leadership styles (transformational, transactional, and laissez faire) and the prevention of adolescence pregnancy among university students at Universidad César Vallejo, in Peru.\n\n\nMethods\n\nWe performed a non-experimental, quantitative, cross-sectional study, with a correlational descriptive design.\n\nThe study was conducted from June 8, 2018 to July 20, 2018. Questionnaires were handed to students every Friday. Survey questions were administered in the classrooms, on the Universidad César Vallejo campus, a subsidiary on the Tarapoto campus. Average time of 25 min was allowed for completing the questionnaires.\n\nTo strive for completeness and diversity, we invited all students in person during classes enrolled in the starting two semesters at Universidad César Vallejo, Peru. Students were only included if they (1) agreed to participate in the study voluntarily; and (2) had physical and mental capacity to answer the questionnaires.\n\nOur sample population consisted of 793 students, which equaled all students within the eligibility criteria.\n\nOur questionnaire described below was validated by exploring (i) ACE (Adverse childhood experiences), including possible emotional, physical, or sexual abuse experienced at the age of less than 18 years; this was tested in a randomized manner, on 40 students (this number represents 5% of the total number of students tested in our study); next a (ii) preliminary pilot testing was performed by engaging 80 students (this number represents 10% of the total number of students tested in our study), and finally, (iii) reliability testing was conducted on internal consistency, test-retest, and inter-rater. Result of our preliminary testing validated the survey, such that no changes had to be implemented.\n\nThe questionnaire comprised three sections: (I) General demographic data, (II) Teacher leadership styles, and (III) Pregnancy prevention teachings. Sections II and III had a rating scale from 1 to 5, according to the Likert scale (Boone & Boone, 2012). A copy of the questionnaire can be retrieved from the Extended data (Contreras & Lowy, 2020).\n\nSection II contained 17 questions for transformational leadership, 14 questions for transactional leadership, and 9 for laissez faire leadership, to sum to a total of 40 questions. Based on results of section II, teaching leadership styles were grouped into three categories according to Vásquez (2012), namely (i) Transformational (high control and low acceptance), (ii) Transactional (high control and high acceptance), and (iii) Laissez faire (low control and high acceptance) (Table 1).\n\nSection III addressed 10 questions on primary prevention, 5 questions on secondary prevention, and 4 questions on tertiary prevention. Answers allowed to assess, whether the teacher provided low, medium, or high level teaching for each type of prevention, as follows: for > 63, high teaching level was assigned; at ≤ 63-42, medium teaching level was considered; while < 42 corresponded to low teaching level .\n\nWe applied Chi-Square for statistical analysis, using SPSS (version 26) to reveal possible correlations between teaching leadership styles and prevention of adolescence pregnancy. Significant correlations were accepted at the p <0.05 level, as widely applied in public health-related studies.\n\nThe committee of the Vicerrectorado de Investigación, Universidad César Vallejo approved this study (021-2018-VI-UCV) on April 16, 2018. All participants were informed on the scope and content of the survey, on their rights as participants, and for additional information and possible questions, they were provided the name and electronic address of a designated contact person. All participants signed a consent form.\n\n\nResults\n\nParticipant characteristics are listed in Table 2.\n\nBased on the survey test results, there is no significant difference between the distribution of the various teaching leadership styles, e.g., according to students’ insight, the transformational, transactional, and laissez daire styles were 50.4, 51.5, and 55.5, respectively, at high perspective level (Table 3).\n\nWe found that the types of pregnancy prevention, used by students were the following: 44.4% apply “high” level of “primary prevention”, 36.6% “medium” level of “tertiary prevention,” while 36.2% utilize a “low” level of “secondary prevention” (Table 4).\n\nVia the non-parametric Chi square test, we determined that leadership styles transformational, Transactional, and Laissez Faire significantly impact the prevention of pregnancy (X2 = 136.390, p = 0.0000; X2 = 95.539, p = 0.0000; and X2 = 80.557, p = 0.0000, respectively) (Table 5). We evidenced a substantial correlation between the leadership variable and prevention of pregnancy of higher education students (X2 = 110.634; p = 0.0000 <0.05). Our results agree with findings by Jordán & Blanco (2015), i.e., that health education is unsatisfactory in the higher education system.\n\n\nDiscussion\n\nLeadership implies an influential relationship between two or more people; therefore, the leadership of teachers constitutes a fundamental axis in education, training, and personal-social development of college students. Classroom education is triggered by a series of interactions between teachers and students, among students, and between diverse groups of students, all these collaborations being guided by rules of coexistence. Overall, these processes enable learning and integral development of students. Also important is the effectiveness of teachers in creating an appropriate climate for learning and collaboration.\n\nDirect relationship between teaching leadership and coexistence sets the necessary characteristics for reducing pregnancy of college students and by this, to avoid consequences of desertion, low learning, discrimination, and more importantly, to prevent perinatal maternal morbidity and mortality. Our study documents the significant interconnection between leadership variables and pregnancy prevention of higher education students. Teaching leadership has been demonstrated as a key factor in pregnancy prevention, regardless of leadership style. From students’ perspective, the leadership style of university teachers is rated as 55.5% high level Laissez Faire leadership. Regarding types of pregnancy prevention, we found that 44.4% are a result of teachers high level instruction of primary prevention.\n\nLimitations and bias may be related to answers provided by college students in the presence of their teacher, or because of misinterpretation/misunderstanding of some questions. This inconvenience was avoided by allowing students to answer questions in the absence of the teacher and providing the research team’s assistance in clarifying doubts or mistakes that may arise. Hence, the research group contributed to elucidate unclear situations that occur during the survey. Directors of César Vallejo University authorized the development of the study and approved the time for its application.\n\nResults can be considered as part of a general situation that exists at different universities of the country, as students starting their studies have the highest dropout rate, caused by pregnancy. In addition, the teacher is not only the facilitator of learning, but also a dynamic person, who exercises leadership by advising, mentoring, and guiding university students toward sustainable learning. Teachers are engaged active human beings, who can make positive decisions on students’ lives.\n\n\nConclusions\n\nBased on our statistical analysis, one can conclude that for college students, teaching leadership style significantly affects the prevention of adolescent pregnancy. We believe that this is an important finding, which complements a prior study (Jordán & Blanco, 2015) that focused on the importance of health education in the higher education system.\n\n\nData availability\n\nFigshare: Datasets, https://doi.org/10.6084/m9.figshare.13385612.v1 (Contreras, 2020).\n\nFigshare: Survey, https://doi.org/10.6084/m9.figshare.13385405.v1 (Contreras & Lowy, 2020).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgments\n\nThe authors express special thanks to all members of the Genesis Sustainable Future, Ltd., Sárospatak, Hungary, for their assistance in manuscript preparation and statistical analysis.\n\n\nReferences\n\nAnzoategui J: Plan de Intervención Educativo para la Prevención de Embarazo en la Adolescencia. El Centro de Tesis, Documentos, Publicaciones y Recursos Educativos más amplio de la Red. Términos y Condiciones. 2015; (Accessed: January 21, 2021.). Reference Source\n\nArroyave K, Cuartas Y, García J: Concepciones sobre la maternidad y paternidad en adolescentes de la Institución Educativa Jaime Salazar Robledo. Colombia. 2015; 7–8. (Accessed on January15, 2021). Reference Source\n\nBoone HN, Boone DA: Analyzing likert data. J of extension. 2012; 50(2): 1–5. Reference Source\n\nContreras: Datasets. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.13385612.v1\n\nContreras, Lowy DA: Survey. figshare. Journal contribution. 2020. http://www.doi.org/10.6084/m9.figshare.13385405.v1\n\nJordán Padrón M, Blanco Pereira ME: Educación sanitaria integral del adolescente en el Instituto Preuniversitario Urbano José Luis Dubrocq, de Matanzas. Revista Médica Electrónica. 2015; 37(3): 197–206. Reference Source\n\nNewman K, Harrison L, Dashiff C, et al.: Relaciones entre los tipos de padres y comportamientos de riesgo en la salud del adolescente: una revisión bibliográfica integrada. Rev Latino-América Enfermagem. 2008; 16(1): 142–150. Publisher Full Text\n\nRamírez E, Contreras G: Las competencias docentes de los profesores del nivel medio superior sobre el tema de sexualidad y el VIH/SIDA. (Tesis de licenciatura). Universidad Autónoma del Estado de México. México. 2013. 2013; (Accessed: January 6, 2021.). Reference Source\n\nReátegui B, Carranza K: Influencia del liderazgo docente en la prevención del embarazo adolescente en estudiantes del 5to año de secundaria de la Institución Educativa “Francisco Izquierdo Ríos”, distrito Morales, región San Martín, año 2016. (Tesis de licenciatura). Universidad Nacional de San Martín Tarapoto. Perú. Recuperado de. 2016; (Accessed: November 7, 2020.). Reference Source\n\nRosario L: Estilos de liderazgo de los maestros y maestras de salud escolar del nivel secundario en la implantación del programa de educación en salud escolar. Universidad del Turabo”. (Ph.D. Dissertation). Escuela de Educación. Puerto Rico. 2012; (Accessed: December 12, 2020.).\n\nUNICEF: Vivencias y relatos sobre el embarazo en adolescentes. Una aproximación a los factores culturales, sociales y emocionales a partir de un estudio en seis países de la región informe final. Oficina Regional para América Latina y el Caribe. Panamá. 2014; (Accessed: January 18, 2021.). Reference Source\n\nVásquez D: Estilos de liderazgo de los docentes del nivel secundario de una institución educativa del distrito de la Perla-Callao. (Tesis de maestría). Universidad San Ignacio de Loyola. Facultad de Educación Programa Académico de Maestría en Educación para Docentes de la Región Callao. Lima Perú. 2012. Reference Source" }	[ { "id": "128635", "date": "14 Apr 2022", "name": "Seyed Ali Azin", "expertise": [ "Reviewer Expertise Social Medicine/Sexual Health", "Sexual Medicine" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAlthough the result of the research seems logical, the article has many ambiguities:\nTeaching leadership styles are described very briefly. Also, \"Types of pregnancy prevention\" is not clear at all. In both cases, if the reader does not refer to the text of the questionnaire, he/she cannot get a clear picture of the concepts stated in the article; for instance, in public health discourse, primary pregnancy prevention often refers to the use of contraceptives.\n\nWhy and how was the ACE questionnaire used to develop and validate the research tool?\n\nThe content of the tables also does not help much to clear up the ambiguity. For example, the basis of the cut-off points for the \"teaching level\" score is not clear.\nOverall, it seems that in order to communicate better with the reader, the article needs to be seriously revised.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-239
https://f1000research.com/articles/10-238/v1	24 Mar 21	{ "type": "Research Article", "title": "How to dismantle modern stressors: does a short trip to simulated Paleolithic conditions in the wild reduce cortisol levels?", "authors": [ "Jens Freese", "Simon Schnell", "Alina Schäfer", "Rainer J. Klement", "Sascha Krüger", "Laura Lückemann", "Helmut Lötzerich", "Simon Schnell", "Alina Schäfer", "Rainer J. Klement", "Sascha Krüger", "Laura Lückemann", "Helmut Lötzerich" ], "abstract": "Background: Chronic stress has become a central problem of our modern society. It appears plausible that our Paleolithic ancestors were more exposed to acutely life-threatening stress stimuli than chronically enduring psychosocial stressors. The aim of this study was to test whether it is possible to reduce physiological and psychological stress parameters by returning to simulated Paleolithic lifestyle conditions. Methods: In total, 15 volunteers undertook a four-day hiking tour simulating hunter-gatherer lifestyle conditions in a National Park. Saliva samples were taken at 5, 6 and 7 pm prior to, during and after the intervention and evaluated for cortisol concentration. Perceived stress was measured by the Perceived Stress Questionnaire. Results: The study was completed by 11 of the subjects. Mean body weight and fat mass decreased significantly during the intervention by 2.3 and 2.2 kg, respectively. There was a marked increase in cortisol levels on the first day of the intervention compared to baseline with a gradual return to baseline levels on the subsequent days. Individual cortisol responses were heterogeneous; in threes subjects, cortisol concentrations generally decreased from 5 to 7 pm, while in four subjects the opposite trend was observed.\n\nConclusions: During this four-day outdoor intervention under simulated Paleolithic conditions, significant changes occurred almost exclusively at the first day of the study. The increase in cortisol levels at this point supports the hypothesis that some individuals respond by an increased release of cortisol as a result of adaptation to new environmental conditions.", "keywords": [ "Cortisol", "Exercise", "Paleolithic diet", "Stress" ], "content": "Introduction\n\nOver the past 2.5 million years, humans, and the species preceding them, have continuously adapted to the corresponding environmental conditions1. For the longest period of this development, humans subsisted through a hunter-gatherer lifestyle2. However, environmental conditions changed ~12,000 years ago with the cultivation of plants and domestication of animals, and even more drastically ~150 years ago with the beginning of industrialization and 30 years ago with the advent of digitization3,4.\n\nAdaptation to stress signals is an integral part of human evolution5. The concept of the stress response is based on the idea that organisms activate protective mechanisms that allow them to survive unfamiliar and potentially dangerous situations6. Those who are resilient to internal and external danger signals increase their chance of survival, thereby also ensuring the survival of their genes7.\n\nChronic stress has become a central problem of our modern society, which is associated with numerous diseases, such as obesity, type 2 diabetes or depression8. It appears plausible that our Paleolithic ancestors were more exposed to acutely life-threatening stress stimuli than chronically enduring psychosocial stressors such as existential anxiety; indeed, many modern-day stressors including pressure to meet certain deadlines, social media stress etc. are a very recent phenomenon. Here, we report findings of a study that was designed to subject civilized humans to an imitation of the Paleolithic lifestyle for a short period of time. The research question was whether it is possible to reduce physiological and psychological stress parameters by returning to simulated Paleolithic conditions in a National Park over a four-day duration.\n\n\nMethods\n\nThe study was approved as a natural study by the ethics committee of the German Sports University Cologne (061-2018). All subjects signed a written informed consent concerning possible risks of participation before the start of the intervention. For a realistic simulation of Paleolithic conditions, the following basic requirements were defined:\n\n1. During the intervention, subjects should remain exclusively in the wild.\n\n2. In order to simulate hunting and gathering behavior, subjects should travel a distance of at least 20 km per day.\n\n3. The first meal was not to be consumed before 12 am to simulate a period of hunting and gathering.\n\n4. The diet should be based on the principles of a “Paleolithic diet” as defined by Cordain9, including lean meats, fish, fruits, vegetables, nuts, oils (coconut oil, olive oil) and sweet potatoes.\n\n5. The sleep-wake cycle should be adapted to the natural circadian rhythm according to sunrise and sunset.\n\nThe study lasted a total of five days and four nights from 25th to 29th July 2018. However, the last day was for return only, so the intervention spanned a total of four full days. The tour was led by Sascha Krüger (SK), who himself was a participant in our pilot studies published by Freese et al.3,4, and offers these tours commercially under the name \"BACK2BASIC-Tour\". Our pilot studies took place in 2015 and 2016. The general study design was the same as in this follow-up study, but the research objective had focused on metabolic parameters.\n\nAt the beginning of the intervention, a 30-minute information session was held by the study director. Subjects were given extensive information about the exact procedure of the study as well as their tasks and were given the opportunity to receive answers to their open questions. During the entire intervention period, the study management team was available on site as scientific back-up. To analyze stress levels, saliva samples were taken before (t0), during (t1-4) and after (t5, t7, t11) the intervention to determine cortisol levels.\n\nAll participants in the commercially offered BACK2BASIC tour were informed in advance about our study, and written informed consent was obtained. Exclusion criteria were any known diseases or the administration of medications that could affect cortisol levels. Participants completed a 30-minute on-site introductory seminar about the main principles and goals of the study.\n\nTo simulate Paleolithic conditions, the following interventions were implemented during the study period:\n\nProvision of “Paleolithic foods”: At lunchtime participants received a food package consisting of two pieces of fruit and vegetables and 100 grams of nuts. Before sunset, we delivered a freshly prepared, Paleo-compliant meal to their place of residence.\n\nSpending 24 hours each day in an open arboreous environment\n\nCut off from technology and modern-style work stress (e.g., notifications from mobile phones, email, time pressure, etc.)\n\nExposure to the natural 24-hour temperature variability (only modest amount of clothing and sleeping bag was allowed)\n\nHiking for several hours each day starting after sunrise\n\nThe subjects were within the group at all times, supervised by the experienced tour guide SK. Simon Schnell (SS) was present each day at dinner to collect the saliva samples and hand out the new ones for the following day. Otherwise, there were no contacts during the interaction. Since subjects were constantly outside, all participants inevitably had to adhere to the study guidelines.\n\nHealth Questionnaire and Anthropometric Data. Prior to the start of the intervention, each subject completed a health questionnaire that collected the following parameters: Personal, anthropometric and physical activity data, dietary behavior, health complaints and diseases. In addition to the requested anthropometric data, body weight, fat-free mass, skeletal muscle mass, body fat mass, and body mass index (BMI) were determined using a bioimpedance scale (Tanita model DC 430 MA P). At the end of the intervention, measurements on the bioimpedance scale were repeated. A copy of the questionnaire is provided as extended data10.\n\nSaliva samples. Three days before, during, and seven days after the 4-day intervention (i.e., from t0–t11), three saliva samples were collected from each subject at 5, 6, and 7 pm, respectively. Saliva samples were collected in Salivettes Cortisol Code Blue (Sarstedt, Nümbrecht, Germany) and analyzed for cortisol concentrations in the laboratory of the Institute of Medical Psychology and Behavioral Immunobiology, Universtity Hospital Essen, using a commercially available enzyme-linked immunosorbent assay (Cortisol ELISA, IBL International, Hamburg, Germany) according to the manufacturer’s instructions as previously described11,12. Intra- and inter-assay variance was 4.8% and 5.9% respectively, the detection limit was 0.005 μg/dL.\n\nPsychological Stress Parameters. Perceived stress was measured every evening at the time of saliva sampling by the German version of the Perceived Stress Questionnaire13. The PSQ contains 20 items and comprises four subscales (worries, tension, joy and demands). The questionnaire’s instruction asked the participants to rate how often an item applied to their life within the last four weeks or last four days, respectively. The rating scale ranges from 1 (“almost”) to 4 (“usually”). An overall score of perceived stress can be built by a summation of the four subscales.\n\nMood was assessed with the help of the German version of the Multidimensional Mood Questionnaire14. The Multidimensional Mood Questionnaire contains 12 items and comprises three mood dimensions (pleasant–unpleasant, awake–sleepy, and calm–restless). The questionnaire’s instruction asked participants to rate different adjectives regarding their current mood.\n\nPhysical measurements. Temperature was measured by the tour guide using a standard temperature meter. Daily hiking distance was measured by the smartphone app “Komoot – Cycling & Hiking Maps”.\n\nStatistical analysis. The Shapiro-Wilk test was used to test for normally distributed anthropometric parameters and cortisol values. The pre-post intervention comparison was carried out by means of a dependent t-test for normally distributed variables, whereas differences between non-normally distributed pre-post parameters were checked for significance by means of the Wilcoxon rank sum test. Because of the lack of normal distribution, analysis of variance of the repeatedly measured cortisol data was performed using the Friedman test. In order to compare the individual intervention days, a post hoc test was used. Significance values were adjusted by Bonferroni correction. Parameters with p-value <0.005 were considered significant15, p <0.001 highly significant. We used Microsoft Excel 365 and IBM SPSS Statistics (version 25) for analysis.\n\nA pre-post intervention comparison of the psychological stress parameters (perceived stress and mood) was conducted using dependent t-tests.\n\n\nResults\n\nOut of 15 recruited volunteers, 11 completed the study (7 female and 4 male). Two were unable to attend for organizational reasons, while two others left during the intervention due to illness. The mean age of those who completed the study was 40.9 years (range 27–59 years). None of the participants was on medical treatment and all were considered mentally and physically healthy.\n\nDue to experiences of our studies from 2013–2014, we did not measure the average kcal intake per day and the macronutrient composition during the four nights and days in the wild. Average temperature was 33° Celsius at day one. On the other days, average temperatures ranged between 26–29° Celsius. The average daily hiking distance was 25.3 km.\n\nFour days of simulated Paleolithic conditions resulted in significant changes in subjects' body composition. Body weight dropped on average from 78.5 kg to 76.2 kg (- 2.9%, p = 1.185 × 10-13). Body fat mass was reduced from 20.8 kg to 18.6 kg (- 2.3 kg, p = 0.0002), which corresponds to a reduction of the body fat percentage from 26.3% to 24.2% (- 2.1%). All changes in body composition were highly significant (see Table 110).\n\nP-values have been Bonferroni-corrected.\n\nFriedman's multi-factorial analysis of variance confirmed that there were differences in cortisol levels between the individual measurement days (p = 7.19 × 10-16). As a result, post-hoc tests were performed taking the time of sampling into account. An initial analysis of average cortisol levels throughout the day (see Figure 1) revealed a marked increase in salivary cortisol at t1 with a slight increase to t4. As it turned out, nearly all significant changes in cortisol levels were related to t1. Within 12 cases of significant changes, t1 was involved ten times.\n\nFigure 1 shows cortisol levels over the individual measurement days, according to the time of sampling. The curve at 5 pm shows at t1 a lower increase in cortisol compared to 6 pm and 7 pm (p=0.00146 and p=0.000706, respectively). Nevertheless, cortisol level at 5 pm displays a significant difference between t1 and t7 and between t4 and t7.\n\nThe mean cortisol level at t1 increased almost threefold from 5 to 6 pm (from 10.82 to 29.5 ng/ml). The cortisol value at 6 pm on the first day (t1) differed significantly from all other 6 pm cortisol values, except on t4. Consistent with the general trend, the 6 pm cortisol curve shows a significant increase from t0 to t1, followed by a rapid drop at t2. On the other hand, the slight increase in salivary cortisol at t4 does not differ significantly from the measurements on the other days at 6 pm.\n\nAt 7 pm, the highest cortisol levels of the study were measured at t1 (mean 34.83 ng/ml, maximum 170 ng/ml). Most of the significant changes at 7 pm are related to t1. In addition, 7 pm cortisol levels differ significantly from t4 to t7, as in the measurements at 5 pm. Regardless of sample-taking time, there were no significant differences in the pre-post comparison. Neither at t5, t7 nor t11 significant changes from the initial value at t0 could be found. The only significant changes from t0 occurred at t1. Even during the intervention itself, there were hardly any significant changes in cortisol levels. Only between t1 and t3 (6 pm) and between t1 and t2 (7 pm) cortisol levels decreased significantly.\n\nTo investigate differences in cortisol values between 5–6 pm, 6–7 pm and 5–7 pm, a variance analysis was performed. The outcome was positive (p=7.19×10-16). Post-hoc tests showed significant differences in cortisol levels over several days, depending on the time of the day, but not on all days. At t0, t1, t2, t3 and t11, cortisol changed significantly in the course of time. In contrast, there were no significant differences in cortisol measured at the different times of the day at t4, t5 and t7.\n\nIn two out of eight days, there was a significant change in cortisol levels between 5 and 6 pm (at t1 and t3). Between 6 pm and 7 pm cortisol levels did not vary significantly. Most of the changes were detected between 5 and 7 pm (at t0, t1, t2, t3 and t11), especially during the intervention period lasting from t1–t4.\n\nAs Figure 1 shows, cortisol levels decreased from 5 to 6 pm and from 6 to 7 pm on six out of eight days, except for t1 and t4. Nevertheless, as shown in Figure 2, mean salivary cortisol levels showed a continuous increase from 5–6 pm (4.32 ng/ml to 5.9 ng/ml) and 6–7 pm (5.9 ng/ml to 6.33 ng/ml).\n\nCortisol levels were also examined individually in all subjects. For this purpose, subjects were divided into “pro”-responders and non-responders. The former were comprised of subjects whose cortisol levels generally decreased from 5 to 7 pm (on all days), whereas subjects whose cortisol levels generally increased from 5 to 7 pm were rated as non-responders. In total, three subjects were classified as pro-responders, four subjects as non-responders. Noteworthy, cortisol levels of pro-responders increased significantly at the beginning of the study (see Figure 3). At baseline (t0), the average concentration of salivary cortisol was 8.27 ng/ml at 5 pm, 5.25 ng/ml at 6 pm and 4.01 ng/ml at 7 pm. Non-responders had already low cortisol levels at the beginning of the study (see Figure 4).\n\nIn a pre-post comparison, pro-responders achieved a significant reduction in cortisol levels. From t0 to t11, average cortisol decreased from 8.27 to 2.18 ng/ml at 5 pm, from 5.25 to 1.37 ng/ml at 6 pm, and from 4.01 to 1.95 ng/ml at 7 pm. In contrast, cortisol levels of non-responders increased between t0 and t11, albeit to a lesser extent than pro-responders decreased. Four subjects could not be classified as pro- or non-responders. These neither had a consistent increase nor a reduction of cortisol levels in the pre-post comparison.\n\nAnalyses showed a trend for a change regarding the overall stress score (t (9) = 2.45, p = 0.04) and the perceived worries (t (9) = 2.51, p = 0.03) and demands (t (9) = 3.03, p = 0.01) from pre to post intervention.\n\nNo significant changes concerning participants’ mood were found from pre to post intervention (pleasant–unpleasant: t (9) = 0.20, p = 0.85, awake–sleepy: t (9) = 0.63, p = 0.55, and calm–restless: t (9) = -1.05, p = 0.32).\n\n\nDiscussion\n\nThe aim of our study was to test how living in the wild for a short period of time affects markers of stress, which is an important research question given that nowadays many people spend significant amounts of time in highly artificial environments.\n\nDaily hiking tours averaging 25.3 km per day exposed subjects to physical stress. Research has shown that medium to high intensity exercise (60-80% VO2max) leads to an increased cortisol output, whereas moderate exercise (40% VO2max) reduces cortisol16. Walking is generally classified as a low-intensity exercise. However, due to the long hiking distances and the uncommon high ambient temperatures, individual activity levels may have been strenuous for most of the subjects. Brenner et al.17 revealed a cumulative effect of moderate- to high-intensity exercise and heat on cortisol output. In addition, they pointed out that repetitive physical stress at high temperature conditions causes elevated stress reactions, which last for longer periods after cessation of exercise. This outcome may explain the sharp and progressive increase of cortisol levels at t1.\n\nDue to the high ambient temperature and the above-average intensity for most subjects on the first day of the intervention, it should be considered that the large increase in cortisol levels at t1 may be due to synergistic effects of heat and exercise. In three of 11 subjects, cortisol levels at t1 showed no changes at all. Obviously, these subjects may be accustomed to prolonged stress in the heat. Health questionnaires confirmed this assumption: two of these subjects regularly participated in similar events, especially the tour guide who completed this intervention for the third time with other groups that year. One subject regularly performed endurance training. According to Martikainen et al.18 endurance training results in a reduced activity of the hypothalamus-pituitary-adrenal (HPA) axis with correspondingly lower cortisol release. For all other subjects, who were not accustomed to the extensive physical workload in combination with heat and intermittent fasting conditions, the cumulative lifestyle changes would likely have been a strong stressor.\n\n\nConclusions\n\nDuring this four-day outdoor intervention under simulated Paleolithic conditions, significant changes occurred almost exclusively at the first day of the study (t1). The increase in cortisol levels at this point supports the hypothesis that non-responders respond by an increased release of cortisol as a result of adaptation to new environmental conditions. Although we could not find an average pre-post change in this small sample size, the individual analysis shows encouraging results. The distinction between pro- and contra-responders displays that subjects with high psychosocial stress could benefit from such an intervention. Future studies should recruit a larger number of subjects with more relevant inclusion criteria, such as subjects, who are permanently exposed to high levels of psychosocial stress.\n\nAs a limitation of this study, all analyses should be considered with caution, due to the small sample size and heterogeneity of the study participants.\n\n\nData availability\n\nZenodo: How to dismantle modern stressors: does a short trip to simulated Paleolithic conditions in the wild reduce cortisol levels? (Study data). http://doi.org/10.5281/zenodo.454951610\n\nThis project contains the following underlying data:\n\n- Distance.png\n\n- Confirmation Ethics Application Eifel Study 2018.pdf\n\n- Cortisol Evaluation 26.7. with Time Comparison per Day.spv\n\n- Output Body Data.spv\n\n- Evaluation Questionnaires Eifel Study.sav\n\n- Data Sheet Body Data.sav\n\nZenodo: How to dismantle modern stressors: does a short trip to simulated Paleolithic conditions in the wild reduce cortisol levels? (Study data). http://doi.org/10.5281/zenodo.454951610\n\nThis project contains the following underlying data:\n\n- Health Questionaire & Informed Consent.pdf\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgments\n\nWe would like to acknowledge Alexandra Korowski from the Institute of Medical Psychology and Behavioral Immunobiology (Essen, Germany), who analyzed cortisol samples and helped with data entry.\n\n\nReferences\n\nCordain L, Eaton SB, Sebastian A, et al.: Origins and evolution of the Western diet: health implications for the 21st century. Am J Clin Nutr. 2005; 81(2): 341–354. PubMed Abstract \| Publisher Full Text\n\nO´Keefe JH, Vogel R, Lavie CJ, et al.: Exercise Like a Hunter-Gatherer: A Prescription for Organic Physical Fitness. Prog Cardiovasc Dis. 2011; 53(6): 471–479. PubMed Abstract \| Publisher Full Text\n\nFreese J, Ruiz-Núñez B, Heynck R, et al.: To restore health, “Do we have to go back to the future?” The impact of a 4-day Paleolithic lifestyle change on human metabolism – a pilot study. J Evol Health. 2016; 2(1). Publisher Full Text\n\nFreese J, Pardi D, Ruiz-Nunez B, et al.: Back to the future. Metabolic effects of a 4-day outdoor trip under simulated Paleolithic conditions - new insights from the Eifel study. J Evol Health. 2016; 1(1). Publisher Full Text\n\nArnoldini M, Mostowy R, Bonhoeffer S, et al.: Evolution of Stress Response in the Face of Unreliable Environmental Signals. PLoS Comput Biol. 2012; 8(8): e1002627. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFerenci T: Maintaining a healthy SPANC balance through regulatory and mutational adaptation. Mol Microbiol. 2005; 57(1): 1–8. PubMed Abstract \| Publisher Full Text\n\nVia S, Gomulkiewicz R, De Jong G, et al.: Adaptive phenotypic plasticity: consensus and controversy. Trends Ecol Evol. 1995; 10(5): 212–217. PubMed Abstract \| Publisher Full Text\n\nBadura B, Ducki A, Schröder H, et al.: Fehlzeiten-Report 2015: Neue Wege für mehr Gesundheit-Qualitätsstandards für ein zielgruppenspezifisches Gesundheitsmanagement. Springer-Verlag. 2015. Publisher Full Text\n\nCordain L: Die Paleo-Ernährung. Das (r)evolutionäre Ernährungskonzept für Fitness, Gesundheit und Gewichtsmanagement. Köln: Deutscher Trainer Verlag. 2013.\n\nFreese J, Schnell S, Schäfer A, et al.: How to dismantle modern stressors: does a short trip to simulated Paleolithic conditions in the wild reduce cortisol levels? (Study data) [Data set]. Zenodo. 2021. http://www.doi.org/10.5281/zenodo.4549516\n\nPetrakova L, Doering BK, Vits S, et al.: Psychosocial stress increases salivary alpha-amylase activity independently from plasma noradrenaline levels. PLoS One. 2015; 10(8): e0134561. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPetrakova L, Boy K, Mittmann L, et al.: Salivary alpha-amylase and noradrenaline responses to corticotropin-releasing hormone administration in humans. Biol Psychol. 2017; 127: 34–39. PubMed Abstract \| Publisher Full Text\n\nFliege H, Rose M, Arck P, et al.: The Perceived Stress Questionnaire (PSQ) Reconsidered: Validation and reference values from different clinical and healthy adult samples. Psychosom Med. 2005; 67(1): 78–88. PubMed Abstract \| Publisher Full Text\n\nSteyer R, Schwenkmezger P, Notz P, et al.: Der Mehrdimensionale Befindlichkeitsfragebogen (MDBF). Göttingen: Hogrefe.1997. Reference Source\n\nBenjamin DL, Berger JO, Johannesson M, et al.: Redefine statistical significance. Nat Hum Behav. 2018; 2(1): 6–10. PubMed Abstract \| Publisher Full Text\n\nHill EE, Zack E, Battaglini C, et al.: Exercise and circulating cortisol levels: the intensity threshold effect. J Endocrinol Invest. 2008; 31(7): 587–591. PubMed Abstract \| Publisher Full Text\n\nBrenner I, Shek PN, Zamecnik J, et al.: Stress hormones and the immunological responses to heat and exercise. Int J Sports Med. 1998; 19(2): 130–143. PubMed Abstract \| Publisher Full Text\n\nMartikainen S, Pesonen AK, Lahti J, et al.: Higher Levels of Physical Activity Are Associated With Lower Hypothalamic-Pituitary-Adrenocortical Axis Reactivity to Psychosocial Stress in Children. J Clin Endocrinol Metab. 2013; 98(4): 619–627. PubMed Abstract \| Publisher Full Text" }	[ { "id": "82127", "date": "20 Apr 2021", "name": "Julia Otten", "expertise": [ "Reviewer Expertise clinical trials", "statistics", "nutrition", "diabetes" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this paper, Freese and colleagues describe weight loss and afternoon salivary cortisol levels before, during and after a 4-day hiking tour. This 4-day activity was supposed to simulate Paleolithic conditions with a walking distance of at least 20 km per day, cut off from technology, exposure to the natural 24-hour temperature variability and a Paleolithic diet with the first meal consumed at 12 a.m. at the earliest. This 4-day intervention caused weight loss and a marked increase in salivary cortisol at the first day of intervention. Salivary cortisol returned to baseline values at day 2, 3 and 4 of the hiking tour. This is a well-conducted small study with interesting results on day 1 of the intervention. However, the statistical presentation of the results (see 4 and 5 below) needs to be improved until the paper can be recommended to a wider audience.\nCould the authors describe the rational why they decided to measure cortisol at 5, 6 and 7 p.m.? In my opinion, these are quite unusual time points. It would therefore be important to know the reason for this choice of time points.\n\nIn this paper, higher salivary cortisol levels at day 1 of the hiking tour compared to before the tour but also compared to day 2 of the hiking tour are found. This finding is discussed very well in the paper. However, also cortisol levels at 6 p.m. and 7 p.m. at day 1 are significantly higher than the cortisol levels at 5 p.m. at the same day. I suggest that the authors discuss possible explanations for this finding. To find an explanation, it would be important to know what study participants did during 5 p.m. and 7 p.m. at day 1.\n\nThe authors describe in the methods that they used the Wilcoxon rank sum test to compare the non-normally distributed pre- and post-test variables. However, the Wilcoxon rank sum test is usually used to compare two independent groups which is not the case in this study. I would suggest using the Wilcoxon signed rank test instead.\n\nThe file with the original data is published together with the paper. Because of the very low p-values in table 1, I calculated all statistical test for table 1 and I have to state that all p-values in table 1 are wrong. Maybe the authors applied the Bonferroni correction in the wrong direction? An easy way to apply the Bonferroni correction: You decide for which number of analyses you want to correct for; some authors choose to correct for the number of analyses in one table, other authors correct for the number of analyses in the whole paper. In this paper, table 1 has four analyses, so the Bonferroni corrected significance value is p = 0.05/4 = 0.0125. Comparing weight before and after the intervention with an independent t-test results in p = 4.26 x 10-7. So, weight loss is significant in this study because this p-value is below the significance value of p = 0.0125.\n\nI would suggest adding a measurement of variability to all figures, e.g. standard deviation, standard error of the mean or 95% confidence intervals.\n\nWhenever exact numbers are depicted in the text of the results section or the abstract, a measurement of variability, e.g. standard deviation, should be added. However, if the exact numbers are described in the table, you do not need to repeat those numbers in the text of the results section.\n\nIt should be added to the figure legend of table 1 that ‘mean ± standard deviation’ is depicted in this table. In table 1, the standard deviation should be added for the difference.\n\nBecause of the significant weight loss in this study, it would be valuable to report calorie intake.\n\nSeveral participants left the intervention early. Are there weight and cortisol measurements available for those participants? Did those participants differ from the other participants that completed the intervention?\n\nIt is highly debated if salivary cortisol is a good biomarker for stress; for chronic psychological stress it may not be a biomarker at all. The authors should add a couple of sentences about the usefulness of their biomarker to the discussion section. An example of a paper that discusses this: Hellhammer DH et al: Salivary cortisol as a biomarker in stress research. Psychoneuroendocrinology (2009) 34, 163-1711.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "92095", "date": "09 Sep 2021", "name": "Terry Wahls", "expertise": [ "Reviewer Expertise Dietary intervention studies of the modified paleolithic diet in the setting of multiple sclerosis" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nHow to dismantle modern stressors: does a short trip to simulated Paleolithic conditions in the wild reduce cortisol levels? [version 1; peer review: 1 not approved]\nThe article investigates the effect of 4 days of hiking approximately 25 km/ day in a forest setting. Paleo style snacks and a paleo meal are provided. Salivary cortisol is obtained at 5, 6, and 7 pm and analyzed. Cortisol is elevated most significantly at 5 pm on the first day but is also elevated at 6 and 7. Over time the cortisol elevations are lessened. Psychological stress parameters lessened over time, no significant change in mood over time was observed.\nIntroduction\nWhat were your aims and hypotheses?\n\nWhat were the eligibility inclusion/ exclusion criteria?\n\nRecruitment strategy?\nMethods\nItem #3 first meal not be consumed before 12 am is probably a typographical error.\n\nWhat foods were provided in the paleo compliant meal?\n\nWere people allowed to eat additional foods ad libitum?\n\nWhere was the residence?\n\nThe participants usual residence or with the group in tents, shelters, cabins, hotels, or their homes?\n\nWere these foods a change from the usual dietary intake of participants from your health questionnaire? Please describe as a table.\n\nResults\nReasons for dropouts?\n\nPlease provide demographic information from your health questionnaire.\n\nPlease provide the perceived stress, multidimensional mood questionnaire score data as a table, mean SD pre and post, change value and P value in the text or in a table.\n\nDid the cortisol response differ according to the prior exercise level or dietary pattern? Define how you determined pro responder and non-responder. Identify this as a post hoc analysis.\nI am willing to review after revisions.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-238
https://f1000research.com/articles/9-1380/v1	27 Nov 20	{ "type": "Software Tool Article", "title": "DEVILS: a tool for the visualization of large datasets with a high dynamic range", "authors": [ "Romain Guiet", "Olivier Burri", "Nicolas Chiaruttini", "Olivier Hagens", "Arne Seitz", "Romain Guiet", "Olivier Burri", "Nicolas Chiaruttini", "Olivier Hagens" ], "abstract": "The number of grey values that can be displayed on monitors and be processed by the human eye is smaller than the dynamic range of image-based sensors. This makes the visualization of such data a challenge, especially with specimens where small dim structures are equally important as large bright ones, or whenever variations in intensity, such as non-homogeneous staining efficiencies or light depth penetration, becomes an issue. While simple intensity display mappings are easily possible, these fail to provide a one-shot observation that can display objects of varying intensities. In order to facilitate the visualization-based analysis of large volumetric datasets, we developed an easy-to-use ImageJ plugin enabling the compressed display of features within several magnitudes of intensities. The Display Enhancement for Visual Inspection of Large Stacks plugin (DEVILS) homogenizes the intensities by using a combination of local and global pixel operations to allow for high and low intensities to be visible simultaneously to the human eye. The plugin is based on a single, intuitively understandable parameter, features a preview mode, and uses parallelization to process multiple image planes. As output, the plugin is capable of producing a BigDataViewer-compatible dataset for fast visualization. We demonstrate the utility of the plugin for large volumetric image data.", "keywords": [ "Large datasets", "ImageJ/Fiji", "Image Processing", "BigDataViewer", "Light-sheet fluorescence microscopy" ], "content": "Introduction\n\nThe display of data from a light microscope is challenging as the modulation transfer function, which dictates the contrast, is size dependent. Images of small objects have intrinsically a lower contrast than larger ones. In particular, objects with a size close to the resolution limit of the imaging system are hardly contrasted, especially when imaged simultaneously with larger objects (Figure S1, see Extended data (Guiet et al., 2020a)). This intrinsic drawback is even reinforced by the fact that the labelling efficiency might not be uniform for small and large structures. Moreover, optical aberrations, in particular spherical ones, further degrade the signal in a non-linear and sample-dependent manner due to refractive index mismatches. The visual inspection of such image data is therefore challenging. This is often the case for large volumetric datasets as typically provided by light-sheet fluorescence microscopy (LSFM).\n\nIn LSFM, detection and image acquisition of the images is camera based, delivering images with a bit depth of 12 bits and beyond. The resulting image stacks can therefore exhibit a dynamic range that is larger than the grey values that can be distinguished by the human visual system or that can be displayed on monitors and screens.\n\nFiji is one of the mostly used open source software for biological image data (Schindelin et al., 2012). However, it struggles with the display and rapid inspection of large volumetric datasets with a high dynamic range. We therefore propose a Fiji plugin that facilitates on-screen display of structures with intensities differing by several orders of magnitude called Display Enhancement for Visual Inspection of Large Stacks (DEVILS).\n\nDEVILS performs local tone-mapping of fluorescence microscopy images, which has been commonly applied in every day world (Salih et al., 2012) and medical image processing (Park & Montag, 2007). To achieve this in fluorescence images from biological samples, DEVILS uses a combination of three image processing routines. The original image is divided by a processed copy of itself (i.e. convolved with a Gaussian kernel) followed by a nonlinear intensity modification (square root operation) and a local rolling-ball background subtraction. Together they are implemented as a Fiji plugin. To deal with the size of the datasets, in the range of hundreds of gigabytes, the plugin can work on multiple planes of a virtual stack in parallel threads.\n\nWe compare this workflow with several well-known and established methods to adjust the display of an image and furthermore analyse the effect of the DEVILS algorithm by comparing objects varying in intensity and size, as well as the different noise and background levels in the image. Details of the workflow and its implementation as a user-friendly ImageJ plugin are discussed afterwards.\n\n\nMethods\n\nCleared right murine midbrain, using active clarity protocol (Lee et al., 2016) (Figure 1 and Figure 3). Multichannel stacks were acquired as tiles with 15% overlap on a Zeiss Lightsheet Z1 using a 20×/1.0 clearing objective. Each channel was acquired sequentially for each slice using single-sided illumination and a lightsheet thickness of 5.5 μm at the center and an optic zoom of 0.75×. The channels were acquired in the following order: tdTomato, endogenous staining of dopaminergic neurons (Ex: 561 nm, Em: BP 575 – 615 nm); Alexa Fluor 488, immunostaining of TH (Ex: 488 nm, Em: BP 525 – 545 nm) and DAPI, nuclear counterstain (Ex: 405 nm, Em: 420 – 470 nm). Original acquisitions yielded voxel sizes of 0.29 μm in XY and 1.5 μm in Z. The resulting tiles were fused and downscaled to an isotropic resolution of 1.2 μm using BigStitcher (Hörl et al., 2019).\n\nAn optical slice of a chemically cleared mouse midbrain was imaged with light-sheet fluorescence microscopy (LSFM) and different image processing operations are compared. Images are displayed using the Fire look-up table (LUT) except for A which is displayed in grey-scale. Apart from panels A, B and D, the auto contrast (AC) adjustment of Fiji was set to 0.35. (A) Unprocessed image displayed using a grey LUT with the minimum (min) and maximum (max) display settings set to the pixel histogram values (568 – 55656). Scale bar: 100 µm. (B) Unprocessed image displayed using the Fire LUT with the same display settings as in A. (C) Unprocessed image displayed using AC adjustment. (D) Unprocessed image displayed with min-max set to 568 – 3709 (3709 equals 1/15th of the maximal intensity). (E) Original image displayed using Gamma adjustment (=2). (F) Original image after applying the Square Root operation. (G) Original image after a division by a spatially filtered version of the image (Gaussian blur, =50). (H) Original image after applying the ‘Subtract Background’ method from Fiji/ (radius = 25 px). (I) Original image after applying the DEVILS plugin with p = 25.\n\nCleared whole mouse (Parvalbumin-Cre C57BL/6) brain using CLARITY1 protocol (Figures 5A and 5B). Single channel stack acquisition on a mesoSPIM system (Voigt et al., 2019) (Wyss Center Advanced Light-sheet Imaging Center, Geneva, Switzerland) using a 42 Olympus MVX-10 zoom macroscope with a 1× objective (Olympus MVPLAPO 1×), for a final magnification of 0.8× for Figure 5A and 2.0× for Figure 5B. Stacks were acquired in single-sided illumination. The acquired channel represents the expression of php.eB AAV (Chan et al., 2017) through endogenous expression of TdTomato (Ex: 561 nm, EM: LP 561 nm, BrightLine HC, AHF). Final voxel sizes are 8.23 μm in XY and 5 μm in Z for Figure 5A, and 3.26 μm in XY, and 3 μm in Z for Figure 5B. pAAV-FLEX-tdTomato was a gift from Edward Boyden (Addgene plasmid # 28306 ; http://n2t.net/addgene:28306 ; RRID:Addgene_28306)\n\nDrosophila larva brain (Figure 5C). The sample was dissected in PBS and suspended in a 2 mm diameter capillary with 1% agarose and imaged on a Zeiss Lightsheet Z1 using a 20×/1.0 water objective. A single channel with dual-sided illumination was acquired in GFP over a Z-stack. This acquisition was repeated over four angles (multiview acquisition) at 90° intervals. The acquired channel represents endogenous expression of nuclear GFP on a subset of motoneurons (Ex: 488 nm Em: BP 525 – 545 nm). Original acquisitions yielded voxel sizes of 0.46 μm in XY and 1.16 μm in Z. The resulting multiview acquisition was registered and fused to an isotropic voxel size of 0.46 μm using BigStitcher (Hörl et al., 2019).\n\nOne of the benefits of LSFM is that large specimens can be imaged at subcellular resolution. However, the modulation contrast obtained with diffraction limited light microscopy scales with the size of the imaged objects. An object with a size equal to the diffraction limit of the imaging system is half as bright compared to objects with two times the size of the diffraction limit. The intensity of objects with sizes half the diffraction limit is a mere 10% compared to the larger objects mentioned earlier (Williams & Becklund, 2002). Adding variation of biomarker expression and staining efficiency to the equation makes it clear that images of specimens with both large and small objects will contain areas whose intensity differs by several orders of magnitude. Brain tissue constitutes a prominent example (see Figure 1). Regions with clusters of cells with high pixel intensities and rather dim individual cells typically make displaying the image even more challenging. In the following section, we will compare common display modifications and outline the DEVILS workflow.\n\nIn Figure 1 an optical slice of a chemically cleared part of a mouse brain is displayed, acquired with a LSFM. Meaningful inspection of the dataset requires use of the entire dynamic range of the camera throughout the acquisition and its processing for inspection. Figure 1A displays the image data using a grey-scale lookup table (LUT), using the minimum and maximum pixel value as the upper and the lower limit for the display mapping function. This procedure is not giving satisfactory results as only the bright structures in the lower right corner are visible.\n\nBetter visualisation of image data is obtained with a coloured LUT as the human eye can distinguish more colours than grey scales. This standard approach exploits the 24 bits of information (RGB colour) to render a wide range of intensities of a single channel visible (Silva et al., 2011). Figure 1B shows the application of the “Fire” LUT from Fiji, using the minimum and the maximum pixel value as the upper and the lower limit for the display mapping function. Comparison with Figure 1A reveals that faint structures can be better recognized in the coloured image. However, the improvement is not sufficient to display all image data. Figure 1C and 1D illustrate that more structures become visible when adjusting the linear display function. These linear display adjustments combined with coloured LUTs are still not sufficient to display all of the image information: structures that are not visible in the top right corner of the image in Figure 1B and 1C become visible in Figure 1D when the maximum of the display function is lowered. However, clipping artefacts are visible at the same time for the brightest structures in the middle of the image.\n\nAnother well-known approach to improve visualization is the application of non-linear display functions. The most common one is gamma correction, which modifies the display exponentially using a value ɣ as the exponent of the original pixel intensity. Using ɣ >1 facilitates the recognition of bright structures. Thus, it can be used to suppress a homogeneous background in the sample (e.g. out of focus light, autofluorescence or unspecific antibody staining). However, it is of note that dim structures disappear (Figure 1E). To help visualise dim structures, ɣ <1 is better suited. For example, using ɣ = 0.5, mathematically identical to calculating the square root of the image, helps to identify faint objects in the top right part of the image (Figure 1F). Note that fainter objects are easier to identify than in the aforementioned images using linear display settings (Figure 1A – D).\n\nA popular method to remove intensity values from large objects is to divide the image by its blurred version. The resulting image of such an operation is shown in Figure 1G. The operation acts as a high-pass filter. It requires careful selection of the width of the gaussian filter and can lead to artefacts on the edge of objects, as well as an increase of the background signal and of the noise in the faintest areas of the original image. Using a so-called ‘Subtract Background’ method (aka rolling ball method) one can avoid such undesired perception effects (Figure 1H) but this does not help much with reducing the range between the low and high intensity values.\n\nTherefore, we propose here a simple workflow that homogenizes the intensities and removes background so that high and low intensities are visible simultaneously for the human eye. It requires one parameter p, the pixel size of the object of interest. The obtained result is the image Figure 1I that allows the observer to visualize low and high intensity objects without further adjustments.\n\nOur current implementation of the aforementioned workflow is a Fiji plugin that uses a simple image processor and parallelizes its processing on the available cores of the machine (Figure 2A). This enables the processing of large image stacks in a reasonable amount of time: it takes seven minutes to process a 12 GB stack on a workstation typically used for image processing. The plugin reads the selected file as a virtual stack and accesses each individual plane for processing. Only one parameter p is needed. It shall correlate with the size of the largest object in the image, in pixels. The plugin performs a division by a Gaussian blurred version of the image (=2p), calculates the square root and subtracts the background using the rolling ball method as implemented in Fiji, with a radius equal to p. The output images are then exported as individual tif files. A virtual stack reconstituted from the individual tif files can be opened with a ‘Open DEVILS Folder’ command. Furthermore, images can also be exported in the Hdf5 format, which allows their inspection with the BigDataViewer (Pietzsch et al., 2015).\n\n(A) Schematic workflow of the DEVILS ImageProcessor (IP) operation and its parallelization in order to handle large image stacks.\n\n(B) Graphical user interface of the plugin. In the standard mode, only the image location and the parameter p is required from the user. The advanced mode enables the user to define the output directory, define an object size per channel, minimum and maximum values for conversion (necessary with 8-bit and 16-bit output) and the output bit depth (8-, 16- or 32-bit).\n\n(C) Graphical user interface of the preview plugin.\n\nAll parameters are entered via a graphical user interface (GUI) as shown in Figure 2B and are recordable as a macro. The advanced parameter options allow the user to specify the output folder, change the output bit-depth of the images (8-, 16- or 32-bit) and specify the minimum and maximum ranges for conversion to an 8-bit or 16-bit image. Such a conversion decreases the size of the output data but requires careful selection of the minimum and maximum values to avoid data clipping artefacts. In the basic mode, a minimum and maximum of -100 and 10000, respectively, are set and the image bit depth is fixed at 16-bit. For rapid testing of the DEVILS operation, a preview mode is available (Figure 2C). It processes a single image and can be used for parameter optimization e.g. to find suitable values for the image conversion.\n\nA machine with 8GB of RAM or above will be able to run DEVILS, provided it has enough RAM to contain a single XY plane of your data at any time. DEVILS requires Fiji to run, and can be installed by checking the PTBIOP update site under Help > Update > Manage Update Sites (https://imagej.net/Update_Sites).\n\nTo better understand the effects of the DEVILS operation on image data, we applied it to a synthetic image containing disks with increasing intensities (5 – 250) and with increasing diameters (7 – 50 pixels) (Figure 3A). A background of 2 intensity units was added and the entire signal was subjected to Gaussian noise with a standard deviation of 0.5. Despite the use of the “Fire” LUT, it is impossible to observe the smallest and faintest spots in the original 8-bit image. The intensity profiles for each row of disks with varying diameter are plotted in Figure 3B using different colours (smaller diameter in red, larger diameter in violet). The profiles are similar with intensities increasing from left to right independent of their respective diameters. Similar results are obtained for vertical intensity profiles (Figure 3C). The intensity of the disks is independent of its size.\n\n(A) Synthetic image of disks with increasing pixel intensity values from left to right (5, 10, 20, 50, 100, 150, 250) and of increasing diameter from top to bottom (7 px, 9 px, 11 px, 15 px, 20 px, 25 px, 50 px). Display settings are set to the minimum and maximum intensity values of the image and the “Fire” LUT is used for display. (B) Vertical line profiles over disks shown in A. The different diameters are labelled with different colours. (C) Horizontal line profiles over disks shown in A. the different intensities are labelled with different colours. (D) Image A processed with the DEVILS plugin (p = 25). (E) Vertical line profiles over disks shown in D. The different diameters are labelled with different colours. (F) Horizontal line profiles over disks shown in D. the different intensities are labelled with different colours.\n\nThe effect of the DEVILS operation is visible in Figure 3D, with the smaller and fainter spots now being visible (top left corner of the image) and the intensity of the larger and brighter spots being compressed. The line profiles in Figure 3E – F reveal that the DEVILS output is scaling with the size and the intensity of the input disk. It becomes obvious that larger disks and brighter disks become dimmer after DEVILS processing. The intensity amplification factor (see Figure S2, Extended data (Guiet et al., 2020a)) is larger for low intensity objects of small size. In the example shown here the ratio of input to output object intensity is around two for the dimmest and smallest object (r = 7, I = 5). It drops to 1.6 for the largest object with the same intensity. It drops more markedly with increasing intensity. It is around one for I = 20 and drops to 0.25 for the smallest disk with the maximum intensity (r = 7, I = 250). The intensity of the largest object (r = 50) is damped by a factor of 0.07. In summary: small, dim objects are becoming brighter, the pixel intensity of large, bright objects is reduced. The maximal amplification difference between the smallest dimmest object and the largest brightest object is 30 in the example shown. This enables the researcher to inspect these objects in one single image. However, it must be stressed that the intensity of the signal can by no means be correlated to the protein, antigen or antibody concentration after the DEVILS algorithm has been applied.\n\nFurthermore, the parameter p needs to be carefully chosen. In case it is smaller than the largest objects, artefacts in these objects can be observed. The line profile after DEVILS treatment (p = 25) of the disks with a diameter of 50 pixels indicate that the intensity inside the disk is lower than at the edges. This effect can be avoided by increasing the parameter p to 50 or above.\n\n\nUse cases\n\nIn Figure 4 the DEVILS algorithm was used in order to facilitate the inspection of a 3D multichannel dataset. Dopaminergic neurons in the mouse midbrain expressing the fluorescent protein tdTomato were immunostained with an α-tyrosine hydroxylase (TH) antibody, visualized with an Alexa Fluor 488 secondary antibody (AF488). DAPI was used as a nuclear counterstain. The algorithm was applied to each channel separately. The overlay of the raw images and the outcome of the DEVILS operation are shown in the main panels of Figure 4A and 4B, respectively. Several observations can be made when comparing cropped regions from raw and processed images (dashed square in Figure 4A – B). First, DEVILS processing reveals a lot of hidden detail; this is visible in all channels, but most marked in the AF488 channel. Second, the intensity drop of individual acquisition tiles towards their edges is reduced after DEVILS processing. Third, a final advantage can be seen in the intensity profile plots along the Z-axis of the image stack: DEVILS processing significantly flattens the bell-shaped curve of the mean intensity per slice (Figure 4D and 4F). This enables a more rapid inspection of the 3D-dataset as no intensity adjustments need to be made when moving between Z-planes. In fact, the entire dataset can now be visualized with the same display settings. The utility of DEVILS is furthermore demonstrated in Figure 5 where it was applied to datasets acquired from different species and different microscope setups.\n\nA part of a cleared mouse midbrain was imaged using light-sheet fluorescence microscopy (LSFM). (A) Raw and merged display of the three individual channels of a single optical slice. The DAPI nuclear counterstain is false coloured in azure, the α-TH staining is shown in chartreuse (AF488) and the dopaminergic neurons in bright pink (tdTomato). The image is assembled from 24 individual tile images. The dashed square indicates a region used for cropping. Crops for each of the individual channels are shown below the main panel. Scale bars: 250 µm. (B) Image A after DEVILS processing (p = 25). (C) Cropped region from A showing the dopaminergic neurons at different Z-positions in the image stack, using the “Fire” look-up table (LUT) and auto contrast adjustment set to 0.35 on slice 280. Scale bar: 250 µm. (D) Mean (black) and maximum (Max, grey) intensities (left and right Y-axes, resp.) of the acquisition channel as shown in panel C per Z-plane (X-axis). (E) Cropped region from B showing the dopaminergic neurons at different Z-positions in the image stack, using the Fire LUT and auto contrast adjustment set to 0.35. (F) Mean (black) and maximum (Max, grey) intensities (left and right Y-axes, resp.) of the acquisition channel as shown in panel E per Z-plane (X-axis).\n\n(A) Cleared mouse brain acquired with a mesoSPIM\\0.8X. Comparison of raw and DEVILS processed images of a Z-color-coded projection using the “Physics” look-up table (LUT). The auto contrast was set on slice 600. Scale bar: 500 µm. (B) Cleared mouse brain acquired with a mesoSPIM\\2.0X. Comparison of raw and DEVILS processed images of a Z-color-coded projection using the “Physics” LUT. The auto contrast was set on slice 600. Scale bar: 500 µm. (C) Drosophila brain acquired with light-sheet fluorescence microscopy (LSFM). Comparison of raw and DEVILS processed images of a Z-color-coded projection using the Physics LUT. The auto contrast was set on slice 380. Scale bar: 50 µm.\n\n\nConclusion\n\nWe presented the image processing routine DEVILS, which is capable of simultaneously displaying structures with intensities differing by several orders of magnitude. The limitations of currently available displays and visualization methods preclude viewing image data with such a dynamic range. DEVILS, implemented as a Fiji/ImageJ Plugin, homogenizes intensity differences and removes global and local background. This approach facilitates the inspection of image data with high and low intensities in a single view while at the same time allowing for the rapid inspection of volumetric 3D datasets.\n\nThe algorithm modifies the pixel intensities in a non-linear way and is dependent on the size of the object, its 2D environment and the intensity itself. Intensity-based interpretation of processed image data is therefore not possible. However, it is a versatile tool for the visual inspection of large volumetric datasets, such as those typically obtained from LSFM. Prompt inspection of these datasets is key when assessing the quality of the data and deciding on further acquisition, processing and analysis strategies. Until now, a rapid method to display data with intensity differences covering several orders of magnitude was missing. DEVILS is providing an easy and straightforward approach to overcome these current limitations.\n\n\nData availability\n\nFigshare: Data: DEVILS: a tool for the visualization of large datasets with a high dynamic range. https://doi.org/10.6084/m9.figshare.c.5197940.v2 (Guiet et al., 2020b)\n\nThis project contains the following underlying data:\n\n- Dataset Figure Fig1, Fig3 (raw data underlying Figures 1 and 4 in czi format)\n\n- Dataset Figure 5a, b (raw data underlying Figure 5A and 5B in czi format)\n\n- Dataset Figure 5c (raw data underlying Figure 5C in czi format)\n\nZenodo: DEVILS: a tool for the visualization of large datasets with a high dynamic range. https://doi.org/10.5281/zenodo.4058414 (Guiet et al., 2020a).\n\nThis project contains the following extended data:\n\n- Extended data.pdf (details of sample preparation and image acquisition for Figures 1, 4 and 5; supplementary figures)\n\n- Workflow description.pdf (workflow describing how to process the provided “.czi” file, with BigStitcher and then with DEVILS)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nSoftware availability\n\nSource code available from: https://github.com/BIOP/ijp-DEVILS\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.4095054 (Guiet et al., 2020c).\n\nLicense: GNU General Public License version 3", "appendix": "Acknowledgements\n\nWe gratefully acknowledge the Advanced Lightsheet Imaging Center (ALICe) of the Wyss Center (Geneva, Switzerland) for image acquisition, Thierry Laroche for help with the Z1 imaging, Sverre Grødem and Marianne Fynn for brain sample preparation, Wei Jiao of the group of Prof. Brian McCabe (EPFL, Lausanne, Switzerland) for providing the Drosophila embryos and the Faculty of Life Sciences at the EPFL for continuous support to the BioImaging & Optics platform (BIOP). This publication was supported by COST Action NEUBIAS (CA15124), funded by COST (European Cooperation in Science and Technology).\n\n\nReferences\n\nChan KY, Jang MJ, Yoo BB, et al.: Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems. Nat Neurosci. 2017; 20(8): 1172–1179. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGuiet R, Burri O, Chiaruttini N, et al.: DEVILS: a tool for the visualization of large datasets with a high dynamic range. Zenodo. 2020a. http://www.doi.org/10.5281/zenodo.4058414\n\nGuiet R, Burri O, Chiaruttini N, et al.: Data: DEVILS: a tool for the visualization of large datasets with a high dynamic range. figshare. Collection. 2020b. https://doi.org/10.6084/m9.figshare.c.5197940.v2\n\nGuiet R, Seitz A, Burri O, et al.: BIOP/ijp-DEVILS: Release for Publication (Version v1.0.2). Zenodo. 2020c. http://www.doi.org/10.5281/zenodo.4095054\n\nHörl D, Rusak FR, Preusser F, et al.: BigStitcher: reconstructing high-resolution image datasets of cleared and expanded samples. Nat Methods. 2019; 16(9): 870–874. PubMed Abstract \| Publisher Full Text\n\nLee E, Choi J, Jo Y, et al.: ACT-PRESTO: Rapid and consistent tissue clearing and labeling method for 3-dimensional (3D) imaging. Sci Rep. 2016; 6: 18631. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPark SH, Montag ED: Evaluating tone mapping algorithms for rendering non-pictorial (scientific) high-dynamic-range images. J Vis Commun Image Represent. 2007; 18(5): 415–428. Publisher Full Text\n\nPietzsch T, Saalfeld S, Preibisch S, et al.: BigDataViewer: visualization and processing for large image data sets. Nat Methods. 2015; 12(6): 481–483. PubMed Abstract \| Publisher Full Text\n\nSalih Y, bt Md-Esa W, Malik AS, et al.: Tone mapping of HDR images: A review. In 2012 4th International Conference on Intelligent and Advanced Systems (ICIAS2012), (Kuala Lumpur, Malaysia: IEEE), 2012; 368–373. Publisher Full Text\n\nSchindelin J, Arganda-Carreras I, Frise E, et al.: Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012; 9(7): 676–682. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSilva S, Santos SB, Madeira J: Using color in visualization: A survey. Comput Graph. 2011; 35(2): 320–333. Publisher Full Text\n\nVoigt FF, Kirschenbaum D, Platonova E, et al.: The mesoSPIM initiative: open-source light-sheet microscopes for imaging cleared tissue. Nat Methods. 2019; 16(11): 1105–1108. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilliams CS, Becklund OA: Introduction to the optical transfer function. (Bellingham, Wash: SPIE Press). 2002. Publisher Full Text" }	[ { "id": "75553", "date": "17 Dec 2020", "name": "Sebastien Tosi", "expertise": [ "Reviewer Expertise Signal processing", "image processing", "computer science" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors introduce an ImageJ plugin to enhance the contrast of small, dim objects in microscopy images without saturating brighter regions. Overall the technique enables a single shot view of complex images with large dynamic range without having to constantly fiddle with the contrast adjustments. The plugin is compatible with large 3D images and it somehow complements existing techniques, but its implementation suffers from several glitches that should be addressed (see Software related issues).\nPotential improvements:\nFor completeness, ImageJ Enhance Local Contrast (CLAHE) with window size adjusted to the size of largest objects should be included to the comparison (Figure 1 and related text).\nSome ImageJ native functions (e.g. subtract background) already leverage multiple cores so it is not clear what advantage brings the thread parallelization described. A speed comparison (with and without parallelization) would help. Also, the hardware used for the test report should be precisely described (especially the number of cores of the workstation).\nIt is not clear why the default minimum grayscale value is set to -100. If this is due to the subtract background performed, I would recommend running it with “Disable Smoothing” and setting the default minimum grayscale value to 0.\nIt is not clear what is the advantage of opening an exported folder with “Open DEVILS folder” instead of importing the image sequence. This should be motivated.\nThe extra parameters of the “DEVILS Preview” dialog box should ideally only appear when ticking “Use advanced parameters” and “Largest_object_size” should not appear twice as this is rather confusing.\nI strongly suggest to better highlight the install procedure from ImageJ update sites both in the manuscript and in GitHub.\nSoftware related issues:\nWhen running “DEVILS Preview”, the images obtained are very dim and their grayscale values do not correspond to the values actually exported when applying DEVILS with the same settings.\n\nI got an error when applying the operation from DEVILS Preview: “Unrecognized command “DEVILS Preview”.\nWording:\nThe words used throughout the text should be more consistent, for instance: “DEVILS Workflow”, “DEVILS Routine”, “DEVILS Algorithm”, “DEVILS operation” or “Slice”, “Plane”, “Z-Plane” should be unified.\nThis sentence should be rephrased: “A popular method to remove intensity values from large objects is to divide the image by its blurred version”.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "6295", "date": "22 Mar 2021", "name": "Arne Seitz", "role": "Author Response", "response": "Dear Sebastien, Thanks for carefully reading our manuscript and your useful comments and suggestions. We are grateful for them and convinced that they help to improve the quality of the publication as well as its understandability as well as the DEVILS plugin. Whenever possible we addressed your points either in the new version of the manuscript or the supplemental material. Please find the answers to the individual points you mentioned in the following: 1. For completeness, ImageJ Enhance Local Contrast (CLAHE) with window size adjusted to the size of largest objects should be included to the comparison (Figure 1 and related text). We have added a comparison with ImageJ enhance Local Contrast (CLAHE) in the supplemental material and are referring to it in the main text. We found that this approach can give equally good results however that the parameter finetuning can be time-consuming. Therefore, we think that DEVILS has some advantages for the visualization of large volumetric datasets. 2. Some ImageJ native functions (e.g. subtract background) already leverage multiple cores so it is not clear what advantage brings the thread parallelization described. A speed comparison (with and without parallelization) would help. Also, the hardware used for the test report should be precisely described (especially the number of cores of the workstation). DEVILS is written so that multiple image planes can be processed in parallel in order to speed up the workflow. We have investigated the effect of further parallelization and added the results to the supplemental material section. 3. It is not clear why the default minimum grayscale value is set to -100. If this is due to the subtract background performed, I would recommend running it with “Disable Smoothing” and setting the default minimum grayscale value to 0. We have taken up the suggestion and evaluated the “Disable Smoothing” option. It modifies the resulting image, however negative values are still present as shown by the macro accessible via this gist link, the macro can also be run with the gist link in ij.imjoy.io 4. It is not clear what is the advantage of opening an exported folder with “Open DEVILS folder” instead of importing the image sequence. This should be motivated. The advantage of the “Open DEVILS folder” is to be able to import processed images as stack or hyperstack directly without the need to specify the order (channels, time, slices). This is in particular useful for multidimensional datasets. We have updated the documentation to explain the advantages of using this folder instead of importing the images manually. https://github.com/BIOP/ijp-DEVILS#opening-tiff-files--opening-devils-folder 5. The extra parameters of the “DEVILS Preview” dialog box should ideally only appear when ticking “Use advanced parameters” and “Largest_object_size” should not appear twice as this is rather confusing. We created two separate commands: DEVILS Preview (Basic) and DEVILS Preview (Advanced) in order to avoid appearing the parameter twice. This helps to avoid the above mentioned confusion. 6. I strongly suggest to better highlight the install procedure from ImageJ update sites both in the manuscript and in GitHub. The documentation is now containing a paragraph about the installation procedure. (https://github.com/BIOP/ijp-DEVILS#installation ). 7. When running “DEVILS Preview”, the images obtained are very dim and their grayscale values do not correspond to the values actually exported when applying DEVILS with the same settings. The preview is interactive, and the “Brightness and Contrast” menu can be used to adjust the min and max display range for each channel. This has been clarified in the documentation. https://github.com/BIOP/ijp-DEVILS#devils-preview--advanced-and https://github.com/BIOP/ijp-DEVILS#minimum-resp-maximum-for-final-conversion-step 8. I got an error when applying the operation from DEVILS Preview: “Unrecognized command “DEVILS Preview”. This bug his has been fixed in the latest version available via the update site. 9. The words used throughout the text should be more consistent, for instance: “DEVILS Workflow”, “DEVILS Routine”, “DEVILS Algorithm”, “DEVILS operation” or “Slice”, “Plane”, “Z-Plane” should be unified. The manuscript was changed by using the term “DEVILS plugin” and avoiding the other ones. 10. This sentence should be rephrased: “A popular method to remove intensity values from large objects is to divide the image by its blurred version”. The sentence was rephrased. Thanks again for your comments and suggestions. Best regards" } ] } ]	1	https://f1000research.com/articles/9-1380
https://f1000research.com/articles/9-1210/v1	08 Oct 20	{ "type": "Software Tool Article", "title": "HPAStainR: a Bioconductor and Shiny app to query protein expression patterns in the Human Protein Atlas", "authors": [ "Tim O. Nieuwenhuis", "Marc K. Halushka", "Marc K. Halushka" ], "abstract": "The Human Protein Atlas is a website of protein expression in human tissues. It is an excellent resource of tissue and cell type protein localization, but only allows the query of a single protein at a time. We introduce HPAStainR as a new Shiny app and Bioconductor/R package used to query the scored staining patterns in the Human Protein Atlas with multiple proteins/genes of interest. This allows the user to determine if an experimentally-generated protein/gene list associates with a particular cell type. We validated the tool using the Panglao Database cell type specific marker genes and a Genotype Expression (GTEx) tissue deconvolution dataset. HPAStainR identified 92% of the Panglao cell types in the top quartile of confidence scores limited to tissue type of origin results. It also appropriately identified the correct cell types from the GTEx dataset. HPAStainR fills a gap in available bioinformatics tools to identify cell type protein expression patterns and can assist in establishing ground truths and exploratory analysis. HPAStainR is available from: https://32tim32.shinyapps.io/HPAStainR/", "keywords": [ "protein staining", "Human Protein Atlas", "marker genes", "marker proteins", "exploratory analysis" ], "content": "Introduction\n\nThe Human Protein Atlas (HPA) has performed immunohistochemistry-based visual proteomics for over 15,313 proteins across 59 tissues. Within each tissue a number of different cell types have been scored for staining patterns by a group of pathologists. Therefore, there is a great amount of visual proteomic data that can be used to classify gene or protein lists into specific cell types1–3. Their website is designed to query one protein of interest at a time and there is no option to query a list of proteins to determine if that protein set is enriched in a particular cell type. This would be a useful feature to take advantage of this robust dataset. Other gene list tools such as Enrichr, which query multiple databases for associations, have not incorporated the HPA protein cell expression dataset into their tools4.\n\nWe introduce HPAStainR (https://32tim32.shinyapps.io/HPAStainR/), a Bioconductor R package and Shiny app developed to query the cell staining database of the HPA. HPAStainR allows a user to input a list of proteins/genes and returns a rank ordered list of cell types that are stained for the input list. HPAStainR is customizable, allowing the user the ability to include cancer or normal tissue data, change the HPA confidence levels, toggle the identification of what proteins from the list were detected, generate a p-value for how many cell type specific proteins are counted for a given cell type, and allow the downloading of the output as a comma separated (csv) file.\n\n\nMethods\n\nThe user interface of Shiny HPAStainR is made of a sidebar where one can input their protein/gene list, along with various options to customize the output of the Shiny app. The main panel consists of two tabs. The first tab is the output tab, where the DataTable from the user’s query is output. The second tab is informational giving the user a list of HPA cell types and how many proteins were tested/histologically scored in a given cell type.\n\nThe HPAStainR package is available on Bioconductor. The package shares all of the same functionality as the Shiny web application, including the ability to run the Shiny app locally and acquire all of the data to do so. This allows HPAStainR to be used as the Shiny app or incorporated into a local R pipeline.\n\nHPAStainR is an online Shiny app5, available at http://shinyapps.io, and as a Bioconductor R Package (https://bioconductor.org/)6 with its source code available on GitHub (https://github.com/tnieuwe/HPAStainR). The function has been tested on R version 3.6.1 and later. Minimal requirements are the same as RStudio’s system requirements [https://bit.ly/2UqwXc6].\n\nInstallation: The installation of the HPAStainR package can be done in R using the following commands:\n\n`if (!requireNamespace(\"BiocManager\", quietly = TRUE))\n\ninstall.packages(\"BiocManager\")`\n\n`BiocManager::install(version='devel')`\n\n`BiocManager::install(\"HPAStainR\")`\n\nThe remote-Shiny web application can be accessed via the following link:\n\nhttps://32tim32.shinyapps.io/HPAStainR/\n\nInput: There are three required R objects for the main HPAStainR function to work and one optional data frame. The first two required objects are the public staining files from the HPA, which can be downloaded using the package and the `HPA_data_downloader` function. The third required input is either a vector of proteins or genes or a character list of proteins separated by a space, comma, or newline to be queried in HPAStainR. The optional data frame, used in the Shiny app version of HPAStainR, is a table that contains the percent of proteins that stained the tissue compared to the number of the proteins evaluated in the tissue, represented in Extended Table 17, which can be generated using the `hpa_summary_maker.R` function. This table demonstrates that not all cell types/tissues have the same number of proteins stained for.\n\nOutput: Either a Shiny DataTable or tibble containing the summarized detection of the input list of proteins or genes for each available cell type customized by the options selected before running the analysis.\n\nStaining was scored by cell type in each tissue by a group of pathologists who rated the intensity in each evaluated cell type as “high, medium, low or not detected.” Not all cell types in all tissues were scored, nor were all cell types consistently evaluated. As a result, there are some caveats in the HPA data that should be noted. The distribution of how many proteins are histologically scored in each of the 137 cell types varies in HPA, such that not all results are equal. The number of proteins scored in cell types ranges from 1 in four substantia nigra cell types to over 17,000 in endometrial glandular cells (Figure 1A; Extended Table 17), impacting how often a protein is detected in a given cell type (Figure 1B). The percent of stained to scored proteins demonstrates an enrichment at both extremes of the distribution (Figure 1C). To highlight this discrepancy in testing, we have made the information in Extended Table 1 as an available tab on the Shiny app.\n\nA. A histogram of the 137 cell types showing the amount of proteins histologically scored in each cell type. Four cell types were evaluated for >15,000 proteins (green line) and 61 for <700 proteins (red line). B. A histogram of the 137 cell types on the amount of proteins that had positive staining in each cell type. C. A histogram of the percent of positively stained proteins to the total amount of histologically scored proteins for the given cell type. The extreme ends of the distribution are populated by samples with less than 700 scored proteins.\n\nThe staining score calculation is an arbitrary measure of how well an input list of proteins are enriched for a particular cell type. A formal equation is below, but briefly, it is calculated based on the frequency and intensity of staining within a given cell type. Staining intensity is a percentage of high, medium, low, and not detected counts. The high percentage is multiplied by a value of 100, the medium percentage by 50, and the low percentage by 25, before adding all the results together to generate the final staining score. While arbitrary, we over-weighted high staining as the IHC was more robust and those proteins may better define the cell type.\n\nThe model for the staining score equation is below where t is the total number of proteins from the list tested in the cell type, h is the number of proteins with high staining in the cell type, m is the number of proteins with medium staining in the cell type, and l is the number of proteins with low staining in the cell type.\n\n\n\nThe confidence score is unique to this paper in its comparison of the Panglao Database (PanglaoDB) cell types, and is a modified version of the staining score adjusting for size of the protein list for each cell type from PanglaoDB. Like the staining score, this score ranges from 0–100. The model for the equation is below, where p is the number of proteins tested, with a max p being 50 (standardizing the score range), and the staining score of the protein list in the cell type is represented by s.\n\n\n\nWhile we utilized all expressed proteins in our staining score, we recognize that some proteins demonstrate cell type enrichment. For this analysis we generated the “enriched-protein p-value” based on a χ2 analysis.\n\nTo calculate the enriched-protein p-value we generated a list of cell type enriched proteins for each level of stringency, low, normal and medium. This was done by calculating a percentage of positively stained to evaluated proteins across each cell type to adjust for protein scoring frequency. This percentage generated our ‘enriched proteins’ list from the top quartile of enriched proteins (the proteins present in <25% of the evaluated cell types. The number of proteins were 3,275, 2,543, and 1,235 for low, normal, and high stringency respectively and 3,818 in cancer) (Extended Tables 2 and 37; Figure 2 and Figure 3). The χ2 analysis was based on the staining presence/absence of ‘enriched proteins’ for a given HPA cell vs presence/absence of proteins from a protein list query. For all experiments in this paper, stringency was set to normal.\n\nA. A boxplot of the number of cell types evaluated per protein in each of the 30 histogram bins. The overall median of cell types is 75 proteins with the 1st and 3rd quartile being 74 and 77 proteins respectively. B. A histogram demonstrating the percent of positive staining cells per protein. Three quality stringencies are given. The 1st quartile lines demonstrate the specificity cut off of the distribution used at each stringency level. As expected, the number of proteins in the 1st quartile increase with lower stringency.\n\nA. A boxplot of the number of cell types evaluated per protein in each of the 30 histogram bins. The overall median of cancer cell types is 203 with the 1st and 3rd quartile of 199 and 207 respectively. A number of outliers with ~2x more cell types evaluated are noted. B. A histogram demonstrating the percent of positive staining cancer cells per protein. This distribution is different from normal tissue as some cancer samples of the same cancer type can positively stain for a protein while other samples will not.\n\nAll code for the package and the analysis can be found on GitHub at https://github.com/tnieuwe/HPAStainR and https://github.com/tnieuwe/HPAstainR_dev_paper, respectively.\n\n\nUse cases\n\nHPAStainR uses the publicly available HPA cell type histologically scored staining data to identify the top cell type matches to a queried protein/gene list. It ranks cell types on a 0 to 100 “staining score” (Figure 4). This score is based on the pathologist annotated staining intensity (high, medium, low) of each protein/gene in the query list for each HPA cell type, as a percent of the total number of proteins/genes queried (see Methods). For example, a query of the pancreatic enzymes PRSS1, PNLIP, and CELA3A, along with the protein PRL, would identify “pancreas exocrine glandular cells” as the top hit with a staining score of 75 due to the high staining intensity in three proteins and negative staining of the fourth protein. The second hit would be the “pituitary gland cells in anterior” due to PRL’s high expression in that cell type (score of 25), followed by “intestinal glandular cells” which only have medium staining of PRSS1 (score of 12.5).\n\nA list of comma, space, or line separated proteins or genes are inputted on the left column. Multiple customizations are available for users below to optimize the search parameters for their query of interest. Data is outputted to the right, and further information about the cell types and how many proteins were histologically scored per cell type are available as a second tab.\n\nTo show the functionality of the Shiny app we applied HPAStainR to the Panglao Database, a hub of community-curated cell type markers from single cell data8. We wanted to investigate how well HPAStainR would mark the cell types based on PanglaoDB’s annotations. We downloaded a tsv file of PanglaoDB’s cell type gene marker data, and parsed it down to only human protein coding marker genes. We assayed 146 human cell types and their 3,661 marker genes through HPAStainR. The number of marker genes per cell type in PanglaoDB are variable, ranging from one marker in trophoblast stem cells to 216 in interneurons. A histogram of markers per cell type showing the distribution can be found in Figure 5.\n\nHistogram of the number of marker genes used to define 142 different Panglao cell types.\n\nTo perform analyses between multiple runs of HPAStainR we generated a “confidence score,” a value (theoretical 0–100) that corrected for the staining score’s determination using an additional feature of how many proteins were evaluated (see Methods). This score weighted cell types with multiple marker proteins staining over cell types with a single or fewer marker proteins. Thus, the confidence score allowed us to rank the cell types based on both staining and depth of data.\n\nHPAStainR is agnostic to the source of a protein/gene list. Therefore, an identification of equivalent cell types across two methods provides strong evidence of HPAStainR’s usefulness. Specific protein lists, corresponding to the 146 cell types were evaluated from PanglaoDB in HPAStainR with the top HPA cell types identified for each. To cover both potential user needs, we included in our PanglaoDB output both the top result of HPAStainR and the top result in the appropriate tissue. The confidence score across these comparisons, generated on HPAStainR data, ranged from 1.5 to 66.75. The results of the 146 cell types were divided into quartiles (Qs) based on the confidence score. The average number of proteins associated with a PanglaoDB cell type used to identify the top HPA cell type strongly correlated with the quartile (76.3; 55.7; 23.9; 7.7 proteins in Q1 to Q4, respectively). In the top quartile of scores, 75% (27/36) of cells matched between PanglaoDB and HPA. Of the nine that were not a perfect match, six matched the top hit when limited by tissue type. Of the remaining Q1 PanglaoDB cell types, liver kupffer cells (a type of macrophage), mesothelial cells, and embryonic stem cells, none had matching cell types in the HPA9.\n\nA subset of this analysis can be seen below in Table 1 with the full results being in Extended Table 47. Results were ranked by confidence score, with a strong correlation of higher confidence scores to more accurate cell type assignments between PanglaoDB and HPA. An interesting example are chondrocytes, where the top stained score (27.25) was to TONSIL squamous epithelial cells and the top tissue specific cell type was SOFT TISSUE - chondrocytes (20.75). In addition to the stain score, HPAStainR provides a p-value (and Holm adjusted p-value) based on a separate metric based on cell type specific/enriched protein expression (see Methods). Although tonsil squamous epithelial cells is the top HPAStainR result, the adjusted enriched protein p-value was 1.0 (nonsignificant) while it was p=0.043 for the chondrocytes, indicating cell-enriched proteins favored the correct match.\n\nBoth the overall top HPAStainR result and a tissue-specific result is given. The “Select Tissues” results are from a search performed for the PanglaoDB cell type only within the matched tissue type (ALL CAPITALIZED) in HPA.\n\nWe then demonstrated the functionality of HPAStainR in bulk datasets. We utilized the variable gene expression data from the Genotype Expression (GTEx) dataset that we had previously uncovered as being driven by variation in pneumocytes or the presence of bronchial epithelium10. There were 33 genes identified in the pneumocyte cluster and 70 genes in the bronchial epithelium cluster. HPAStainR was applied separately to both lists and found the top results to be lung pneumocytes and bronchus respiratory epithelial cells respectively (Figure 6A and 6B; Extended Tables 5 and 67). Therefore, across both single cell and bulk gene expression data, we have identified useful functionality to HPAStainR.\n\nA. Represents the output when a list of genes associated with pneumocytes are used as input. B. Represents the output when a list of genes associated with the bronchial epithelium are used as input.\n\n\nConclusion\n\nHPAStainR fills a small gap in our knowledge base by allowing for the query of gene/protein lists against the cellular protein expression pattern data of HPA. As datasets of single cell RNA sequencing analysis become available, it is useful to have a tool to correlate these individual cellular transcriptomic gene profiles with translated protein expression patterns. We have also shown the tool can recapitulate bulk RNA sequencing findings making it a valuable tool to understand the cellular composition of a sample. The HPA is an excellent resource to observe staining patterns within cells across tissues for proteins of interest. The limitations of the study are the quality of the staining across all HPA tissues and the quality/consistency of the pathology scoring of the tissues11. Both of these may impact the scoring achieved for any given query. HPAStainR is a new valuable resource to accelerate exploratory and ground truth queries in the HPA cell type protein staining data.\n\n\nData availability\n\nThe data from PanglaoDB was downloaded at https://panglaodb.se/markers/PanglaoDB_markers_27_Mar_2020.tsv.gz (last updated March 27th 2020).\n\nHuman Protein Atlas normal tissue and cancer tissue data was acquired from the website: https://www.proteinatlas.org/about/download (last visited March 28th 2020)\n\nHarvard Dataverse: HPAStainR – A Bioconductor and shiny app to query protein expression patterns in the Human Protein Atlas, https://doi.org/10.7910/DVN/CL5ZTA7.\n\nThis project contains the following extended data:\n\nExtended Table 1. The number of proteins evaluated and positively stained for each cell type. For each tissue cell combination the number of proteins being positively scored over the number of times proteins are evaluated. These are categorically grouped on the amount of proteins evaluated.\n\nExtended Table 2. The rarity for proteins in normal tissue across different filters. For each gene in normal tissue that was detected, the percent of how often proteins stained compared to how often they were histologically scored based on three quality filters, low, normal and high. Each protein is also labeled if it is considered rare or not in a given tissue based on if its percentage was in the bottom 1st quartile of the distribution for each quality filter. NA indicates that the protein never reached the threshold to be counted in a given filter level. Proteins that never positively stained were not included.\n\nExtended Table 3. The rarity of proteins in cancer samples. The percent positive staining of 15,301 in cancer cells. The quartile of proteins with the lowest values were indicated as rare. Note, there is no quality filter for cancer thus different cancer samples from the same type of cancer can have different staining patterns.\n\nExtended Table 4. The extended HPAStainR output from Table 1.\n\nExtended Table 5. HPAStainR output of cluster A from McCall et al. The full results of HPAStainR when running cluster A of McCall et al. through the package. Pneumocytes were expected and observed.\n\nExtended Table 6. HPAStainR output of cluster B from McCall et al. The full results of HPAStainR when running cluster B of McCall et al. through the package. Bronchial epithelial cells were expected and observed.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\n\nSoftware availability\n\nSoftware available from: https://32tim32.shinyapps.io/HPAStainR/\n\nBioconductor package available from: https://doi.org/doi:10.18129/B9.bioc.HPAStainR\n\nSource code available from: https://github.com/tnieuwe/HPAStainR\n\nArchived source code as at time of publication: https://doi.org/10.5281/zenodo.404871412.\n\nSoftware license: Artistic-2.0\n\nAnalysis code available from: https://github.com/tnieuwe/HPAstainR_dev_paper\n\nArchived analysis code as at time of publication: https://doi.org/10.5281/zenodo.404870413.\n\nLicense: Artistic-2.0", "appendix": "Acknowledgments\n\nWe thank Matthew N. McCall, Zachary P. Brehm, Stephanie Y. Yang, and Veronica F. Busa for their consultation on the creation of the software.\n\n\nReferences\n\nUhlén M, Fagerberg L, Hallstrom BM, et al.: Proteomics. Tissue-based map of the human proteome. Science. 2015; 347(6220): 1260419. PubMed Abstract \| Publisher Full Text\n\nUhlen M, Zhang C, Lee S, et al.: A pathology atlas of the human cancer transcriptome. Science. 2017; 357(6352): eaan2507. PubMed Abstract \| Publisher Full Text\n\nSjöstedt E, Zhong W, Fagerberg L, et al.: An atlas of the protein-coding genes in the human, pig, and mouse brain. Science. 2020; 367(6482): eaay5947. PubMed Abstract \| Publisher Full Text\n\nKuleshov MV, Jones MR, Rouillard AD, et al.: Enrichr: a comprehensive gene set enrichment analysis web server 2016 update. Nucleic Acids Res. 2016; 44(W1): W90–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChang W, Cheng J, Allaire JJ, et al.: shiny: Web Application Framework for R. 2020. Reference Source\n\nGentleman RC, Carey VJ, Bates DM, et al.: Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 2004; 5(10): R80. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNieuwenhuis OT, Halushka KM: HPAStainR – A Bioconductor and shiny app to query protein expression patterns in the Human Protein Atlas. Harvard Dataverse, V1, UNF:6:o2EDbY39avbmTP9qswinCA== [fileUNF]. 2020. http://www.doi.org/10.7910/DVN/CL5ZTA\n\nFranzén O, Gan LM, Björkegren JLM: PanglaoDB: a web server for exploration of mouse and human single-cell RNA sequencing data. Database (Oxford). 2019; 2019: baz046. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAnene DF, Rosenberg AZ, Kleiner DE, et al.: Utilization of HPASubC for the identification of sinusoid-specific proteins in the liver. J Proteome Res. 2016; 15(5): 1623–9. PubMed Abstract \| Publisher Full Text\n\nMcCall MN, Illei PB, Halushka MK: Complex Sources of Variation in Tissue Expression Data: Analysis of the GTEx Lung Transcriptome. Am J Hum Genet. 2016; 99(3): 624–35. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCornish TC, Chakravarti A, Kapoor A, et al.: HPASubC: A suite of tools for user subclassification of human protein atlas tissue images. J Pathol Inform. 2015; 6: 36. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNieuwenhuis T: tnieuwe/HPAStainR: HPAStainR Dev Release (Version 0.99.2). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.4048714\n\nNieuwenhuis T: tnieuwe/HPAstainR_dev_paper: Release of HPAStainR Analysis repository (Version v1.0.0). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.4048704" }	[ { "id": "73228", "date": "05 Nov 2020", "name": "Mazdak Salavati", "expertise": [ "Reviewer Expertise Genetics and Genomics", "Bioinfomatics", "Cell biology." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript describes a novel tool developed for extending access to the visual proteomics dataset produced by Human Protein Atlas (HPA). The main two features that this shinyApp/R-package is as follows:\nEnabling batch query of gene or protein name lists for cell type composition identification. Use of bulk RNA data (tissues) in order to unravel cellular composition of the starting RNA sample.\n\nAuthors have carried out an external validation with PanglaoDB human cell type dataset in order to confirm the soundness of both staining score and confidence score equations which are largely compatible with HPA cell type groups. They have also studied GTEx RNA-Seq (2 cell types) input gene lists with their pipeline which again was confirm by the top hit returned by the tools (albeit varying Staining score).\n\nThis tool has been developed very thoroughly and with a clear demand in the community at its design. However as highlighted by the authors in the conclusion section, one should approach subjective scored histochemistry obtained datasets always with caution. As the scoring bias introduced by scorers will remain as part of the rank outputs.\n\nSuggestions for the authors:\n\nI would highly recommend to include more data from GTEx tissues and expand the result section of your manuscript.\n\nPerhaps consider a cross validation procedure in the future once more datasets are available for the same cell type. A 5-10 fold cross validation can immensely improve the reliability of the output ranked results.\n\nKnowing a package like EnrichR that covers a variety of GSEA and Pathway enrichment databases, would it make sense to collaborate with their development team to expand functionality for cell type prediction through EnrichR? That's a question or challenge for the authors to answer or decide.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "6447", "date": "22 Mar 2021", "name": "Tim Nieuwenhuis", "role": "Author Response", "response": "Thank you for reviewing our paper, below are our responses to your suggestions: Suggestion 1, Recommend to include more data from GTEx tissues: This tool was generated to specifically analyze GTEx data. We are working on a project to use this tool for a more in-depth analysis of GTEx, similar to the lung paper cited. Suggestion 2, Cross-validation Procedure in the future once more datasets are available: That is an excellent idea. As more datasets become available, we will work to incorporate them into this tool. Suggestion 3, Collaborate with EnrichR: Once HPAStainR package has fully matured through the review process, we will determine if it can be included in the excellent EnrichR tool." } ] }, { "id": "72698", "date": "11 Nov 2020", "name": "Laurent Gatto", "expertise": [ "Reviewer Expertise Computational biology", "proteomics", "genomics", "research software development", "open and reproducible research." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nNieuwenhuis and Halushka describe the HPAStainR package, recently released as part of the Bioconductor project. HPAStainR uses protein immunostraining data from the Human Protein Atlas to asses whether a user-provided list of proteins or genes is associated with a particular cell type.\n## Introduction\nThe authors fail to cite other Biocondcutor packages related to the Human Protein Atlas, namely hpar (in Bioconductor for 8 years) and HPAnalyze (in Bioconductor for 2 years). While the functionalities of these packages are different (but see below), citing similar packages in Bioconductor seems very relevant for an paper in the Bioconductor gateway.\n(Note that I am the author of the hpar package)\n## Methods: Operation\nInstallation instructions should not refer to the development version of the package, but instruct users to install and use the release version. For two reasons: first the development of a package doesn't guarantee any stability, second it puts additional burden on the user to (1) potential have to install the development version of R and (2) end up with installing _all_ Bioconductor development packages. Given that package is released now, the installation instructions should absolutely be updated accordingly.\n\nPlease fix code formatting.\nInput:\n\nThe `HPA_data_downloader()` function is used to download 2 datasets. Note that the 'hpar' package could have been a Bioconductor package to integrate with here, so as to avoid repeated downloading of the data and/or to provide some reproducibility in the analyses (see below for details on this).\n``` > ## Executed on the 10 November at 20:01 > HPA_data <- HPA_data_downloader(tissue_type = 'both', save_file = FALSE) > HPA_out <- HPAStainR(c('PRSS1', 'PNLIP', 'CELA3A', 'PRL'),\n\nHPA_data$hpa_dat,\n\nHPA_data$cancer_dat,\n\n'both') ```\n``` > library(hpar) > packageVersion(\"hpar\") ## ‘1.32.1’ > data(hpaCancer) ## load the data > data(hpaNormalTissue) > all.equal(hpaNormalTissue, HPA_data$hpa_dat) [1] TRUE > all.equal(hpaCancer, HPA_data$cancer_dat, check.attributes = FALSE) ## different colnames only [1] TRUE > ## BUT see below > HPA_out2 <- HPAStainR(c('PRSS1', 'PNLIP', 'CELA3A', 'PRL'),\n\nhpaNormalTissue,\n\nhpaCancer,\n\n'both') ``` There are generally two approaches when it comes to using data from remote resources:\nDownload the data on the fly, which allows to use the very latest version of the data, but at the expense of lack of reproducibility/tracking. Indeed, the results can unexpectedly change from one day to another. This would be to option in the HPAStainR package, as well as other Bioconductor packages such as rols (that queries various ontologies, including GO).\n\nPackaging and versioning data to guarantee tracking and reproducibility of the analysis. This is for instance the option provided by hpar (latest hpar release, version 1.32.1, provides HPA data version 19.3, dated 2020/03/06). Other Bioconductor package that offer this solution are GO.db (package GO), and many other Bioconductor data packages.\nIt would be useful for HPAStainR to make these assumptions explicit and to document how to track results: using hpar or manually (and documenting!) storing the tables downloaded using `HPA_data_downloader()`.\nReturning to the reproducibility of the results, despite identical input data (except for the cancer data column names), it is intriguing that the results aren't identical. There are three cell types that have different p-values/adjusted p-values.\n``` > all.equal(HPA_out, HPA_out2) [1] \"Component “p_val”: 2 string mismatches\"\n\n[2] \"Component “p_val_adj”: 1 string mismatch\" > which(HPA_out$p_val != HPA_out2$p_val) [1] 4 5 > which(HPA_out$p_val_adj != HPA_out2$p_val_adj) [1] 1 > HPA_out[c(1, 4, 5), c(\"cell_type\", \"p_val\", \"p_val_adj\")] # A tibble: 3 x 3\n\ncell_type\n\np_val p_val_adj\n\n1 PANCREAS - exocrine glandular cells <0.005 0.076\n\n2 SMALL INTESTINE - glandular cells\n\n0.37\n\n1.000\n\n3 COLON - glandular cells\n\n0.17\n\n1.000 > HPA_out2[c(1, 4, 5), c(\"cell_type\", \"p_val\", \"p_val_adj\")] # A tibble: 3 x 3\n\ncell_type\n\np_val p_val_adj\n\n1 PANCREAS - exocrine glandular cells <0.005 0.038\n\n2 SMALL INTESTINE - glandular cells\n\n0.34\n\n1.000\n\n3 COLON - glandular cells\n\n0.18\n\n1.000\n\n```\nIt would be interesting for the authors to investigate this, given that the PANCREAS - exocrine glandular cells change from non-significant to significant.\n'hpa_summary_maker.R` must be `HPA_summary_maker()'.\nOutput:\nRunning 'HPAStainR()' as indicated in the man page returns a tibble. It is unclear what the \"Shiny DataTable\" output in the text refers to. I couldn't find any further information in the man page. Did the authors possibly mean the 'shiny_HPAStainR()' function? Anyway, the two function should be mentioned in the manuscript.\n\nRunning that very same example, the output table is incoherent in the mode of the variables: p-values (p_val) and adjusted p-values (p_val_adj) are encoded as characters.\n## HPA data distribution\nThe data discussed in 'HPA data distribution' and available in Extended Table 1 seems to be the same one as returned by the 'HPA_summary_maker()' function. Please mention this explicitly, to allow users to easily generate this table for different data.\n## Confidence score calculation\nIt isn't clear why the PanglaoDB needs an additional confidence score, or why it wouldn't be relevant or useful in other contexts.\n## Cell type enriched p-value\nIt isn't clear what is refereed to by low, normal and medium stringency. Based on the straining score calculation equation, it seems to be related to the low, high, medium staining intensity. Please define the notion of stringency.\n## The Panglao Database\nIn Table 1 and in the 'HPAStainR identified many of the cell types in PanglaoDB' section, the authors make use of the confidence score instead of the p-values to support their validation. Why don't they make use of that p-value, advertised in the previous 'Cell type enriched p-value' section?\n## HPAStainR can help determine cell type populations in bulk RNA sequencing\nThe authors show the first hits, matching the expected cell types. Are there any other cell types that match with an adjusted p-value < 0.05?\n## Software availability\nThe authors mention that the analysis code is available from: https://github.com/tnieuwe/HPAstainR_dev_paper. This repositories however contains a lot of 'old' files ('old_vignette', 'old_files', 'old_versions'), including what appears an old version of the package in 'package_HPAstainR'. Version control is the ideal tool to store and track files over time, and dedicated version can be specifically tagged or released.\nAre these old version relevant? What are the differences with the more recent analyses?\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Partly\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "6448", "date": "22 Mar 2021", "name": "Tim Nieuwenhuis", "role": "Author Response", "response": "We thank you for your time reviewing our package. Below we have individual responses to your queries formatted the same way as your review. ## Introduction Thank you for pointing out our oversight. We have included, in the newest version of the paper, a citation for both hpar and HPAnalyze. We will discuss the incorporation of hpar into our package later in this response. ## Methods Operation: In the new version, we have updated this section to properly reflect the release of HPAStainR and the code, properly formatted, to download said library. Input: HPAStainR’s next release, currently available on the main branch of https://github.com/tnieuwe/HPAStainR and the devel version of Bioconductor, will include a vignette section on how to use hpar’s data in HPAStainR. We are currently keeping the function `HPA_data_downloader()` as its one benefit over hpar is that it gives access to the most recent data if the semi-annual Bioconductor release doesn’t pair with the HPA release. In response to the remote data comment, we have also updated the package (v1.1.4) on the master branch for future release (https://github.com/tnieuwe/HPAStainR). The changes to `HPA_data_downloader()` includes the following: The name of the file and function has changed from `hpa_data_downloader()` to `HPA_data_downloader()` Every time a user downloads and saves the HPA files, the date of the download is provided. To help in version control there are three new parameters that assist in maintaining reproducibility: `version_date_normal`: This parameter allows the user to insert a date string in YYYY-MM-DD format to select a normal tissue file downloaded on the respective date. If a date is not supplied the default argument is “last” which will find the latest version of the file. `version_date_cancer`: The same as version date normal, but for the pathology/cancer file. `force_download`: An argument forcing the re-downloading of files from the HPA website. The purpose of this argument is to allow the user to update their local files, as `HPA_data_downloader()` by default will use local files over downloading said files again. We investigated the incongruency between your run of HPAStainR with hpar and our data, and found the issue was most likely due to the usage of simulated p-values in the chi-square analysis. To fix this, and overall improve the tool, we have changed the base test in HPAStainR() to a Fisher’s Exact Test, which works on the non-parametric data we have. From data not shown here, it does not lengthen the run time of HPAStainR(). Therefore all p-values in the paper have been updated to the results of the Fisher’s Exact Test. Output: We have updated the text to reflect the output of both `HPAStainR()` and `shiny_HPAStainR()` separately. If using the base `HPAStainR ()` function a tibble is returned, while the table returned in the `shiny_HPAStainR` is referred to as a Datatable. The p-values were character values due to the usage of `format.pvalue().` This has been revised and changed to numerical values for the next release. ##HPA data distribution We have also clarified Extended Table 1 and its relationship as the output to the ‘HPA_summary_maker()’ function. ## Confidence Score Calculation The creation of the “confidence scores” was strictly for our PanglaoDB analysis comparison and is not useful outside of validation studies. PanglaoDB had cell types with wildly variable numbers of marker genes. This caused cell types with 1 marker gene that had “high” staining (such as Schwann cells in extended table 4) to become a top hit in PanglaoDB (by staining score), but that does not reveal the accuracy of the tools as the marker genes may just be unique to the cell type in its specific tissue. Therefore we generated the confidence score, which controls and adjusts for the number of marker genes used because having ~40 proteins properly staining is more informative than ~2. The confidence scores are not used in the HPAStainR tool due to the higher consistency of protein staining per cell type, allowing the staining score to be sufficient for ranking. ## Cell type enriched p-value We have added more information explaining how the stringency is based on the “Reliability” column and how each level of stringency functions. The next release of HPAStainR will include this information in the description of the stringency parameter as well. ## The Panglao Database We have revised the table to include p-values of the tissue specificity. The reason we used confidence scores is that the p-values were simulated in the chi-square analysis used in HPAStainR, where there is a smaller range of possible p-values. We felt the confidence score was a better way to order the cell types due to its ability to highlight the staining score while controlling for how many genes each PanglaoDB cell type has. ## HPAStainR can help determine cell type populations in bulk RNA Sequencing In extended table 5 stomach glandular cells have the second-lowest adjusted p-value at 0.306 and in extended table 6 fallopian tube glandular cells, nasopharynx respiratory epithelial cells, endometrium glandular cells, and cervix-uterine glandular cells all have an adjusted p-value <.05. However, the bronchus epithelial cell’s p-value is still the smallest at 1.58x10-21 compared to fallopian tube glandular cells at 3.99x10-19. ## Software availability All “old” versions have been removed and will remain so in the next version." } ] }, { "id": "72699", "date": "11 Jan 2021", "name": "Yasin Kaymaz", "expertise": [ "Reviewer Expertise Bioinformatics particularly in RNA-seq", "single-cell genomics", "and algorithm dev. areas." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAuthors introduce a new R package called HPAStainR for quickly identifying putative cell types given a list of protein or gene names by searching these through the Human Protein Atlas database. HPAStainR has been developed to allow users to query multiple gene/protein names in the HPA database and return an ordered list of cell types for which the query list might be enriched. They also make use of the Panglao single-cell expression database to validate their predictions. The main purpose of the tool addresses a need in the field and should be encouraged. However, I would like to mention some of my concerns below about the basis of the tool and the structure of the manuscript.\n\nIt has been mentioned that there were two main input files from the HPA database which can be downloaded with the ‘HPA_data_downloader’ function. Do you need to download all the data from the HPA for each analysis? Is this really necessary? Can't you do this on the fly?\n\nThe staining score seems to be constructed a bit arbitrarily. Based on staining intensity (high, medium, and low), scores are weighted with some arbitrary constant values and normalized by the total query number. But I wonder if there is any skew in this scoring scheme in case extreme queries are tried, such as all high with many proteins or all low, etc. Especially, given the highly non-uniform scoring distribution in the HPA database (Figure 1).\nConfidence score? Why not combine this equation with the staining score? The point of keeping them as separate metrics seems a bit vague. Also, a bit confusing from the user standpoint.\n\nDo the authors use the cell type enrichment p-value in the enrichment at all? If so, it is not obvious from the text.\nFigure 1, 2, and 3; The actual purpose of these figures should be clearly explained in the main text. It is hard to get it unless staring at them for a while. Also, these first three figures are not directly related to the tool explained here. They are rather some statistics showing the key points of HPA data. I would recommend replacing these with more directly related graphs demonstrating the performance of the HPAStainR and including the current ones as supplemental data.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Partly\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "6449", "date": "22 Mar 2021", "name": "Tim Nieuwenhuis", "role": "Author Response", "response": "Thank you for taking the time to review this paper and the associated package, below is a response to your concerns and the changes we’ve made because of them. 1. … Do you need to download all the data from the HPA for each analysis? Is this really necessary? Can't you do this on the fly? In the current version of `HPA_data_downloader()`, as long as the parameter `save_file` is set to `TRUE`, it will only require you to download the files once. The next release of the package (v1.1.4 available on the main branch at https://github.com/tnieuwe/HPAStainR) includes an updated version of the function that marks the download date of the files, and also allows the user to select which downloaded file they want to use in their analysis for backward reproducibility. 2. The staining score seems to be constructed a bit arbitrarily… We acknowledge the somewhat arbitrary nature of the equation, although it is based on prior histology scoring methods more common in histopathology studies. Additionally, we have generated a distribution of 1,000 HPAStainR results on randomly selected genes, including the top 10 results from HPAStainR and all results, for random gene lists of sizes 10, 25, 50, and 100, these are found as Extended Figures 1 and 2. Our findings show the increased number of genes results in lower staining scores. Regardless, for the top 10 results, the distribution appears to be normal. Analysis of all staining data suggests a right skew. We were unable to create an extreme skew, but we cannot entirely exclude it for some unique queries. 3. Why not combine the confidence score with the staining score? The reason that they are separate is because the confidence score is simply a scaled staining score that is only used for PanglaoDB-HPAStainR analyses. The context we used the confidence score was strictly for testing how well proteins considered marker genes in PanglaoDB mark the equivalent cell types in HPA. This was described in greater detail to reviewer 2’s query. 4. Do the authors use the cell type enrichment pvalue at all? Yes, in the original manuscript we use the p-value at the end of the HPAStainR identified many of the cell types in PanglaoDB section. We also show it in figure 6, and in the updated version of the manuscript, we now include it in table 1. Also, the p-values have changed as the analysis has been updated from a X2 analysis to a Fisher’s Exact Test. 5. The purpose of these figures should be clearly explained in the main text. We have added further information in the figure legends to better clarify what the figures represent. HPAStainR does not have an output that lends itself to a graphical representation, however, we have added two extended figures to the manuscript showing the distribution of the staining score in various random samplings as noted above." } ] } ]	1	https://f1000research.com/articles/9-1210
https://f1000research.com/articles/8-1037/v1	10 Jul 19	{ "type": "Research Article", "title": "Developing a core outcome set for a congenital abnormalities surveillance programme in Rwanda – a Delphi consensus study", "authors": [ "Annette Uwineza", "Aline Muhorakeye", "Janvier Hitayezu", "Peter Thomas Cartledge", "Annette Uwineza", "Aline Muhorakeye", "Janvier Hitayezu" ], "abstract": "Background: In 2015 it was reported that approximately 300,000 newborns die within four weeks of birth every year, worldwide, due to congenital anomalies. This represents approximately 11% of neonatal deaths. This has led scientists, clinicians and public health authorities to establish congenital abnormality registries (CARs). There is currently no CAR in Rwanda. In establishing such a registry, it was determined that the first step was to identify the core outcome set (COS) (or minimal data-set) of variables and outcomes for the registry to ensure that the final results are meaningful and employable. This study aimed to use Delphi consensus methods to identify a methodologically robust COS for a congenital abnormalities surveillance programme in Rwanda. Methods: A three-round, modified Delphi study was undertaken between April and June 2017. Round 1 was a literature and internet search followed by an open and closed question round with experts in Rounds 2 and 3, respectively. Results: An initial draft COS of 136 outcomes was created from a review of 15 African studies and 14 international repository tools including the European Surveillance of Congenital Anomalies and the World Health Organization surveillance guidance. In total, 36 and 34 participants took part in Rounds 2 and 3, respectively. A total of 32 new outcomes were added by participants in Round 2. 103 outcomes met the pre-defined consensus criteria and made up the final COS in Round 3. Conclusions: This is the first core outcome set for a congenital abnormality surveillance programme in an African nation identified in the literature. The next stage is to field-test the surveillance programme using passive case-finding in teaching hospitals in Rwanda.", "keywords": [ "Congenital Abnormalities", "Birth defect", "Population Surveillance", "Core-Outcome Set", "Epidemiologic Methods", "Rwanda" ], "content": "Abbreviations\n\nCARs, congenital abnormality registries; OMET, core outcome measures in effectiveness trials; COS, core outcome set; EUROCAT, European Surveillance of Congenital Anomalies; HRH, human resources for health; ICD, International Statistical Classification of Diseases and Related Health Problems; IRB, institutional review board; LMIC, low and middle-income countries; MeSH, medical subject headings; MoH, Ministry of Health; PI, principle investigator; RPA, Rwandan Pediatric Association; SD, standard deviation; WHO, World Health Organization.\n\n\nIntroduction\n\nCongenital abnormalities are defined as malformations of organs or body parts during development in utero, present at birth and are therefore of prenatal origin1,2. The prognosis of neonates with congenital abnormalities is often poor3. Annually, approximately 300,000 newborns die within four-weeks of birth, worldwide, due to congenital anomalies, representing approximately 11% of neonatal deaths4. The most common congenital abnormalities are congenital heart abnormalities, neural tube defects and Down syndrome4. Causes of congenital abnormalities are genetic, environmental or idiopathic. More than 90% of these newborns are born in low- and middle-income countries (LMICs)5. In resource-limited settings, despite the balance of burden of disease, there is limited epidemiological data about the rate, risk factors and types of congenital abnormalities. In Rwanda, data monitoring is already being undertaken via the Integrated Health Management Information System (HMIS)6. This is a nationwide data-surveillance programme, with health facilities reporting the total number of births with congenital anomalies, but no detail of individual cases. Though a positive start it doesn’t capture rich enough data for meaningful objectives to be met.\n\nWhen epidemiological data has been presented the outcomes described are commonly not consistent. The importance of prevalence of congenital abnormalities has lead scientists and public health authorities to establish congenital abnormality registries (CARs). These surveillance systems based on high-quality epidemiological data are required to identify preventable causes and for policymakers to plan care provisions7. A large number of CARs have already been established, predominantly in high-income countries. The European Surveillance of Congenital Anomalies (EUROCAT) initiative is a good example of this. EUROCAT aims to carry out epidemiologic surveillance of congenital anomalies in Europe8. There are several objectives to EUROCAT initiative, including; i) provision of essential epidemiologic information on congenital anomalies in Europe, ii) facilitating the early warning of teratogenic exposures, iii) evaluating the effectiveness of primary prevention, to assess the impact of developments in prenatal screening, and iv) acting as an information and resource centre regarding clusters or exposures or risk factors for concern8. Finally the EUROCAT initiative aims to act as a catalyst for the setting up of registries throughout Europe collecting comparable, standardized data8. These are admirable objectives for the European continent, and resource-limited settings, with significant burdens of congenital abnormalities, should aim to establish registries and surveillance programmes with equally ambitious goals. The World Health Organization (WHO) have also given high-quality guidance on congenital abnormality surveillance programmes2. This guidance includes similar objectives to EUROCAT and also gives further objectives such as detecting clusters (outbreaks) of congenital anomalies2.\n\nWhen undertaking research or creating registries, the outcome measures should be valid, reliable and feasible9. That is, outcomes should adequately meet the criteria of truth, be sensitive to change and be easily applied and interpreted. They should also be relevant to the setting and the stakeholders who will engage with the data. More emphasis is being placed on ensuring the high quality of outcomes measured in research and surveillance programmes. If research has not been conducted to identify the most appropriate outcomes, several problems may impair the usefulness of the research findings in informing clinical practice. For example, researchers may choose outcomes to suit their own needs, heterogeneous outcomes can impair future synthesis of research findings, and without pre-defined outcomes, it is difficult to know if publishing authors have neglected to include outcomes found in their research10,11.\n\nIt is for these described reasons that many researchers are starting their research and/or registry development with a significant piece of research work to identify the “core outcome set” (COS) in the relevant research field, which may also be known as a “minimal data set”. During this process, a great deal of research energy and time is invested into identifying the variables and outcomes to ensure that the final results of the future research and data-collection are meaningful and employable. There are several considerations regarding the choice of method to use to when developing a COS, which include factors such as the need for methodological rigor in the consensus process, ensuring a diverse range of stakeholder opinions, and finally financial constraints and carbon costs that might limit the practicality of face-to-face meetings10,11. The Delphi technique is a well-respected tool for establishing a COS and is a structured process utilizing a series of ‘rounds' to gather information until group consensus is reached. Each Delphi round employs individuals across diverse geographical locations and diverse areas of expertise. The anonymous nature of the Delphi technique also avoids domination of the consensus process by one or a few experts12.\n\nIn Rwanda there is a long-term goal to develop a Rwandan surveillance program to provide monitoring and epidemiologic data that could be a first step in identifying risk factors and to improve the provision of care of the families of children with congenital abnormalities.\n\nThe WHO have given guidance that the variables to be included in a surveillance programme (registry) may vary, depending on the capacity and resources of the health-care system and surveillance programme2. This Delphi-study aimed to use consensus methods to identify a methodologically robust COS for a Rwandan congenital abnormalities surveillance program. This COS was intended to include risk factors, clinical features, syndromes, and outcomes.\n\n\nMethods\n\nA three-round, modified Delphi-study was undertaken between April and June 2017. Reporting of the study is in accordance with the Sinha and Williamson (COMET, Core Outcome Measures in Effectiveness Trials initiative) checklists for creating a COS using Delphi techniques10,11. No study protocol has previously been published for this study.\n\nAim. Round 1 aimed to produce a draft COS using published resources. This is a common practice in Delphi studies with the justification that the number of possible domains and outcomes to include in the COS of a Congenital Abnormality Surveillance program is substantial. Without providing an initial draft COS to participants the level of recruitment and engagement would be low11.\n\nSearch strategy. A PubMed literature and Google internet search was performed to identify CARs (globally) or epidemiological studies investigating the prevalence and description of congenital abnormalities (African continent). PubMed was searched using Medical Subject Headings (MeSH) keywords and synonyms for: “newborn” AND “congenital abnormalities” AND “developing countries” (see Extended data, Additional Supporting File 1 for a full search strategy13). The search was limited to human studies in the English language with no date limits imposed, the final search being undertaken on 26th January 2017. Epidemiological studies providing a description of all congenital abnormalities of an entire population were included (See Supporting File 1). Articles looking at specific congenital abnormalities alone (e.g. cranial defects, congenital heart defect) were excluded. Google was searched using the above synonyms along with synonyms for “registries”.\n\nObtaining outcomes. When contact details were available in the article, authors of the articles/registries were contacted by email to gain their outcome sets, questionnaires and/or data-collection tools. When contact with the author was not possible the outcome set was extracted from the materials available (i.e. the journal article or website).\n\nCoding. Individual outcomes (e.g. gestation, cleft palate, Pierre Robin, etc.; see Additional file 2) were then coded for content and frequency. The outcome codes were categorized into domains (e.g. maternal details, clinical signs, etc.; see Table 1).\n\nRound 1 consensus. We aimed for a minimum of 15 outcome sets from either published papers or repositories. Consensus was predefined to include any domain or outcome found in two or more of the identified registries or journal articles. These domains and items were then added to the first draft of the COS.\n\nInclusion criteria. Participants were all medically qualified physicians and needed current or previous experience of working in a resource-limited setting, such as Rwanda. All participants needed to have experience of caring for children with congenital abnormalities. Physicians were chosen as Round 1 will have identified existing epidemiological tools, and in Rwanda physicians are likely to be the major stakeholders in collecting and utilizing data.\n\nSampling/enrolment. Recruitment was undertaken from the following sources: i) Rwandan paediatricians (n=53) via the Rwandan Pediatric Association (RPA) records, ii) Rwandan pediatric residents (n=49) via the University of Rwanda (UR) records, iii) International paediatricians previously working on the Human Resources for Health programme14 (n=35) via the Ministry of Health (MoH) records, and iv) Authors of journal articles from Round 1 of the Delphi process (n=15) via the correspondence address. Invitations were sent via email with a link to the questionnaire, which is available as Extended data13.\n\nWe aimed to gain responses from a minimum of 15–30 respondents in each round, which is considered the required number for gaining consensus in Delphi techniques15. The response rate was predicted to be 10–20%. Therefore, 152 participants were invited.\n\nFeedback. Feedback from Round 1 was given to participants in the form of the first draft COS, available as Extended data13.\n\nInstructions to participants. Each domain was presented individually, with its respective outcomes, followed by an open-question; “On reviewing the above outcomes for the domain of <<domain title here>> are there any ADDITIONAL outcomes that you feel that SHOULD be included in the Rwandan Congenital Abnormalities Surveillance Program? (You may list these or write free text, as you wish. You do not need to write a justification of your additions)”. Participants were presented with an open-ended text-box where they could “free text” any additional outcomes to include.\n\nCoding and consensus. Outcomes suggested by participants were coded in Microsoft Excel. Consensus was predefined as a minimum of two independent persons suggesting an outcome prior to it being included in the COS for Round 311. The initial draft outcome set from Round 1 and the new outcomes meeting consensus were combined to create the second draft COS.\n\nInstructions to participants. In Round 3, closed questioning was employed. Each outcome of the second draft COS was presented using a 1–9 point scale, as described by Guyatt and the GRADE development group9,16. Outcomes were presented within their domain with the following instructions: “Where 1–3 are unimportant, 4–6 is ambivalent and 7–9 is important, how important is it that the following are included in the Rwandan Congenital Abnormalities Surveillance Program”.\n\nFeedback. Feedback was given to participants by giving the frequency the outcome was described in Round 1 (as a percentage) or if it was a new addition from Round 2.\n\nConsensus. Consensus for inclusion in the final COS was pre-defined as greater than 70% of participants scoring 7–9 (important) AND less than 15% of participants scoring 1–3 (not important)11.\n\nIn Round 1, outcomes and domains were coded and input into Microsoft Excel. For Rounds 2 and 3, Google Forms was used to administer the questionnaire and collect data. A web link to the Google Forms questionnaire was sent individually to each participant via email. Questionnaires13 for Rounds 2 and 3 were kept open for two weeks. An email reminder was sent once for each round. Participants were reminded of the importance of completing both Rounds 2 and 3 of the Delphi process and asked to complete both rounds. However, because of the fully anonymous nature of responses, non-responders from Round 2 were still invited to Round 3. New participants were not invited between Rounds 2 and 3. Data analysis was undertaken in Microsoft Excel and was descriptive based on the above defined consensus.\n\nThis research proposal was approved by the Institutional Review Board (IRB) at the University of Rwanda (155/CMHS IRB/2017). A statement of consent was included in the email communication with participants. Responding to the questionnaire was deemed as informed consent.\n\n\nResults\n\nWe undertook a modified Delphi-study. A study flow diagram is available in Figure 1).\n\nOur PubMed literature search revealed 545 results. Titles were reviewed along with the abstract, where necessary. We identified 15 relevant journal articles giving descriptions of congenital abnormalities on the African continent and meeting the inclusion criteria (see Additional Supporting File 1). This number of articles was appropriate to deliver consensus on key outcomes for the initial draft COS. Therefore, a further journal/literature search was not performed for studies outside of the African continent. The authors of the included papers were contacted and only one of the authors replied with details of their COS. Therefore, outcomes were extracted from the journal articles.\n\nRegarding existing congenital abnormality surveillance programmes (registries), we were unable to identify any active congenital abnormality repositories/registries from the African continent. We therefore extended our search to other continents. This search revealed 14 repositories: seven from Europe (Belarus = 1, Finland = 1, France = 2, Georgia = 1, Malta = 1, United Kingdom = 1), four from North America (USA = 3, Canada =1) and one from Israel. The included datasets also included the tools from EUROCAT8 and the WHO2 which are the best described guidance on surveillance programmes.\n\nAll outcomes from the 15 journal articles and 14 repositories were coded in Microsoft Excel. From these 29 sources a total of 285 outcomes were identified of which 136 (48%) met our pre-defined criteria for consensus to be included in the first draft of the COS. These outcomes were categorized into 10 domains (Table 1). The consistency of outcome sets from these was low with only 8 of the 285 outcomes (3%) being present in ten or more of the 29 articles/repositories (Additional Supporting file 2). Each of the 285 outcomes was found in an average of 2.7 (9.3%) of the 29 journals/repositories (Standard deviation, SD=2.8). Each of the 136 items included in the first draft COS outcomes were found in a mean of 4.6 (15.8%) of the 29 journals/repositories (Standard deviation = 3.1). The most commonly described outcomes were “spina bifida” and “cleft palate/lip” which were both present in 16 (55%) of the articles/repositories. Each journal/repository described a mean of 27 outcomes (SD=14). Repositories had more outcomes than journal articles with averages of 30 and 24 outcomes respectively.\n\nA total of 37 and 34 participants (Table 2) responded, giving a response rate of 24% and 22% for Rounds 2 and 3, respectively. This exceeded our expected response rate of 10% and resulted in significantly more than the 15–30 participants needed for validity of each round. In Round 2, one participant had never treated children with congenital abnormalities and didn’t meet the inclusion criteria. In total, 73% and 63% of participants treated children with congenital abnormalities either frequently or very frequently. Participants were experienced with a mean of 12 years and 11 years of pediatric experience for Rounds 2 and 3, respectively. Participants included general pediatricians, neonatologists, geneticists, neurologists and pediatric residents.\n\nSD, standard deviation.\n\nQuestionnaires were fully anonymous. Participants did not know the identities of the other individuals in the group, nor did they know the specific answers that any other individual gave. However, year of birth and initials were given by subjects in order to assess attrition rate. Of the respondents from Round 2, 17 (46%) contributed to Round 3, giving an attrition rate of 20 subjects (54%).\n\nOutcomes were presented within their domain on a separate page of the electronic questionnaire. After coding, a total of 219 new outcomes were suggested by participants (Table 3). In total, 62 (28%) of these outcomes were either “general suggestions” or already found in this or another domain. There were therefore 157 genuinely new outcomes. Of the 157 new outcomes, 32 (20%) of these were independently suggested by two or more participants and therefore met the pre-defined definition of consensus and were added to the COS and carried through into the final round of the Delphi process.\n\nThe questionnaire13 was divided into ten sections reflecting the ten domains of the second-draft COS. Outcomes were individually presented in their respective domain. There were 168 outcomes presented for scoring between 1 (non-important) and 9 (important) by participants. In total, 103 outcomes (61%) met the pre-defined consensus criteria to be included in the final COS.\n\nThe final steps were to: i) Ensure outcomes were in appropriate domains, ii) Attach International Statistical Classification of Diseases and Related Health Problems (ICD) coding and naming conventions17 to the outcomes, iii) Place all clinical features (n=52) into anatomical systems, and iv) Alphabetise/order outcomes for ease of use (See Supporting File 3).\n\n\nDiscussion\n\nThis study aimed to create a COS for use in a Rwandan Congenital abnormalities surveillance programme. Using structured consensus methods and taking into account the perspectives of experienced pediatric clinicians, a COS (minimal dataset) of 103 variables and outcomes, within ten domains, has been created.\n\nOur PubMed and internet search identified 29 articles and repositories. There was a large number of excluded descriptive studies of specific abnormality types (e.g. neural tube defects only). The literature search has shown that research studies on congenital abnormalities have been done in Africa but well-structured mechanisms for their surveillance are quasi-absent (i.e. journal articles are available but no repositories/registries were found); this is concerning when one considers the finding that more than 90% of newborns with congenital abnormalities are born in LMICs4,5. These findings support the need for upscaling of surveillance programs on the African continent where congenital abnormalities are responsible for a significant burden of disease.\n\nWith a lack of structured mechanisms or tools to detect and follow-up neonates with congenital abnormalities in Rwanda, it was judged an essential first step to gain consensus from physicians who will case-find regarding which items to be included in the surveillance tool. The Delphi technique was found to be both cost-effective and practical with a methodological rigor to reach a large number of diverse experts and with an advantage of avoiding the negative effects of dominant individuals. Pediatricians are well placed to advise on the development of a surveillance programme for congenital abnormalities since they care for affected neonates and children in their daily practice. In Rounds 2 and 3, we received a higher than anticipated response rate and therefore gained more participants than expected. This was a welcome finding and therefore our results offer consensus of a larger body of experienced clinicians. The participants were also from several different settings giving a broader range of experience. The high response rate is also a sign of how important and relevant participants found the subject and hopefully reflects the \"buy-in\" regarding a future surveillance program.\n\nThe consistency of outcomes described in the journals and repositories in Round 1 was low. Each of the 136 outcomes included in the first-draft COS was found, on average, in only 16% of articles/repositories. This lack of consistency supported the need for this Delphi study to develop a locally relevant COS, such as the one described here. The finding that the 14 repositories held a mean of 30 outcomes suggests that our COS may hold too many outcomes. This may be due to the pre-defined threshold for consensus in Round 3. However, it is interesting to note the high number of new outcomes suggested by participants in Round 2 which could suggest that these additional outcomes are in fact needed.\n\nThe next step in developing the Rwandan CAR is to field test (pilot) the COS in the clinical environment. This is to ensure that completing data-collection using the COS is feasible for phyisicians within day-to-day clinical practice. The WHO guidance on surveillance programmes describes that a programme may be population based or hospital/facility based and can use active or passive case ascertainment (case-finding)2. We intend to use the COS developed in this study to commence passive case ascertainment in teaching hospitals, in order to establish baseline data and feasibility of such a surveillance programme.\n\nStrengths of the study include the fact that the Delphi technique is a commonly used consensus method with several advantages whilst minimizing some of the disadvantages associated with collective decision making, for example, domination by individual interests9. We felt it was a priority to include the variables from EUROCAT8 and the WHO2 guidance on surveillance programmes and have used Delphi methods to build on these data-sets. We used physicians from different settings and different levels of clinical experience to ensure there was no dominance of a particular domain.\n\nLimitations include the fact that our participants were limited to clinicians who had experience of caring for children with congenital abnormalities. Patients or their families, researchers and biostatisticians were not included. It was felt that parents/carers would not have a significant enough understanding of the risks, clinical findings and/or syndromes being assessed. In hindsight, involving collaborators from nursing and allied specialties could have been beneficial as they are often primary caregivers in this setting and therefore will be future stakeholders in future surveillance programmes. Further limitations were the attrition rate from Round 2 to Round 3. The use of a computer-administered questionnaire will have inadvertently excluded less computer literate participants. However, it has been found that participants who are willing to participate in consensus panels are generally representative of their colleagues9. A final limitation is the use of non-African repository data-sets in the first draft. The first draft of the COS included outcomes from settings non-similar to our intended setting.\n\n\nConclusions\n\nThis is the first COS for congenital abnormalities identified in the literature from the African continent. It has been developed for use in Rwanda but could relevant for use in the region and other resource-limited settings. However, each setting should develop their data-set based on the resources available to them and the objectives of the surveillance programme18.\n\n\nData availability\n\nHarvard Dataverse: Delphi Rounds 1 to 3 - Developing a Core Outcome Set for a Congenital Abnormalities Surveillance Programme in Rwanda – a Delphi consensus study. https://doi.org/10.7910/DVN/FEQ9IJ13.\n\nThis project contains the following underlying data:\n\nDelphi Round 1–3 (all) data - f1000 (V1).xls (all data gathered during this study).\n\nRound-1 data in domains (presented in Round-2) .xlsx\n\nHarvard Dataverse: Delphi Rounds 1 to 3 - Developing a Core Outcome Set for a Congenital Abnormalities Surveillance Programme in Rwanda – a Delphi consensus study. https://doi.org/10.7910/DVN/FEQ9IJ13.\n\nThis project contains the following extended data:\n\nAdditional Supporting File 1- Search strategy and repositories/journal articles used in Round-1\n\nAdditional Supporting File 2- Round-3 of Delphi (all outcomes)\n\nAdditional Supporting File 3 - Final COS\n\nAdditional Supporting File 4 - Questionnaires for Round-2 and Round-3\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe acknowledge members of the Pediatric Academic team at the University of Rwanda who reviewed and approved by the study protocol.\n\n\nReferences\n\nNational-Institutes-of-Health: Medical Subject Headings (MeSH) [Internet]. [cited 2018 Oct 16]. Reference Source\n\nWorld Health Organization: Birth defects surveillance: a manual for programme managers [Internet]. 2014; [cited 2019 May 28]. 1–126. Reference Source\n\nKar A: Birth Defects in India: Magnitude, Public Health Impact and Prevention. J Krishna Inst Med Sci Univ. 2014; 3: 7–16. Reference Source\n\nWHO: Factsheet: congenital anomalies [Internet]. 2016; [cited 2017 Sep 28]. Reference Source\n\nChristianson A, Howson CP, Modell B: Global Report on Birth Defects [Internet]. March Dimes Birth Defects Found. 2006; [cited 2017 Sep 28]. Reference Source\n\nRwanda Ministry of Health - Integrated Health Management Information System (HMIS) [Internet]. [cited 2019 May 28]. Reference Source\n\nGupta N, Kabra M, Kapoor S: Establishing National Neonatal Perinatal Database and birth defects registry network - need of the hour! Indian Pediatr. 2014; 51(9): 693–6. PubMed Abstract \| Publisher Full Text\n\nEUROCAT Central Registry: EUROCAT [Internet]. 2016; [cited 2017 May 15].\n\nSchmitt J, Langan S, Stamm T, et al.: Core outcome domains for controlled trials and clinical recordkeeping in eczema: international multiperspective Delphi consensus process. J Invest Dermatol. 2011; 131(3): 623–30. PubMed Abstract \| Publisher Full Text\n\nSinha IP, Smyth RL, Williamson PR: Using the Delphi technique to determine which outcomes to measure in clinical trials: recommendations for the future based on a systematic review of existing studies. PLoS Med. 2011; 8(1): e1000393. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilliamson PR, Altman DG, Blazeby JM, et al.: Developing core outcome sets for clinical trials: issues to consider. Trials. 2012; 13: 132. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBoulkedid R, Abdoul H, Loustau M, et al.: Using and reporting the Delphi method for selecting healthcare quality indicators: a systematic review. PLoS One. 2011; 6(6): e20476. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCartledge P: Delphi Rounds 1 to 3 - Developing a Core Outcome Set for a Congenital Abnormalities Surveillance Programme in Rwanda – a Delphi consensus study. Harvard Dataverse, V1, 2019. http://www.doi.org/10.7910/DVN/FEQ9IJ\n\nBinagwaho A, Kyamanywa P, Farmer PE, et al.: The human resources for health program in Rwanda--new partnership. N Engl J Med. 2010; 369(21): 2054–9. PubMed Abstract \| Publisher Full Text\n\nHsu CC, Sandford BA: The Delphi Technique: Making Sense of Consensus. Practical Assessment Research & Evaluation. Pract Assessment, Res Eval. 2007; 12(10): 1–8. Reference Source\n\nGuyatt GH, Oxman AD, Kunz R, et al.: GRADE guidelines: 2. Framing the question and deciding on important outcomes. J Clin Epidemiol. 2011; 64(4): 395–400. PubMed Abstract \| Publisher Full Text\n\nWHO: ICD-10 Version: 2010 [Internet]. [cited 2017 Jul 13]. Reference Source\n\nSever LE: Guidelines for Conducting Birth Defects Surveillance - Report from the National Birth Defects Prevention Network (NBDPN). [Internet]. 2004; [cited 2019 May 28]. Reference Source" }	[ { "id": "54657", "date": "15 Oct 2019", "name": "Paula R. Williamson", "expertise": [ "Reviewer Expertise Core outcome set development" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article presents the items comprising a minimum data set for a registry of congenital abnormalities in Rwanda, determined through a survey of health professionals. It addresses an important topic, since the set up and maintenance of a registry is no small feat, and increasing the likelihood that key, relevant items are measured is critical. I believe the authors are proposing a ‘minimum data set’ (MDS) rather than a core outcome set (COS). A MDS typically includes a COS plus additional variables (for example baseline, risk factors and treatment variables). Use of the term ‘items’ rather than ‘outcomes’, and ‘MDS’ rather than ‘COS’, throughout would be preferable therefore.\n\nBackground\n\nHow did the 15 African studies reviewed determine items for their registries? This information would be of interest to the reader. Is it clear that they did not use a consensus process, and if not, how did they determine what to measure?\n\nMethods\n\nIn round one, a long list of potential items has been created. This is not usually referred to as a ‘draft COS’ however since it consists of a list of any outcome ever measured by one or more groups rather than being based on any consensus process.\n\nThe list of items is long. Was there any randomisation to address the potential for survey fatigue? Could the authors examine the data for this problem? If not, this should be discussed as a potential limitation.\n\nPre-defining various elements of the process can reduce the potential for bias. Although no study protocol has been published in a journal, might there be one available online? Is there a Research Ethics Committee (REC) application where the design is described or did the authors determine that this work did not require REC approval? If the latter, a statement to this effect, with explanation, should be included.\n\nIt does not appear that an individual participant had the opportunity to review scores from other participants, to reflect on their own view, and then to rescore. The study design appears to be a series of two surveys therefore rather than a Delphi survey. This should be clarified.\n\nResults\n\nThe authors state there was an ineligible participant but continue to include their results. This should be clarified and explained, and the tables amended as appropriate.\n\nThe addition of 32 items is high. What might this imply? How many of these additional items were in the final 103?\n\nThe number of new outcomes suggested in round 2 in the ‘syndrome/diagnosis’ domain is 6 in Table 1 but 8 in Table 3. Please clarify this inconsistency.\n\nTable 2 – please add footnotes to describe the ‘Other’ categories.\n\nTable 3 – please order ‘Patient (infant) details’ first to match Table 1.\n\nThere were 8 items common to the previous registries. Were these 8 in the final set? This would be an interesting discussion point.\n\nDiscussion\n\nWhen referring to the number of ‘outcomes’ in previous registries, is it outcomes or rather items?\n\nThe COS-STAD standards require patients (here parents) to be involved in the determination of important outcomes. Some discussion as to why parents were not involved in this project would be of interest to the reader.\n\nMDS and COS projects often include an in-person meeting to finalise the set. Some discussion as to whether this was considered and, if so, why it was not pursued would be of interest to the reader.\n\nWhy is ‘the use of non-African repository data-sets in the first draft’ necessarily a limitation?\n\nConclusion\n\nThe authors conclude that a new set of registry items should be developed for each setting. I believe this approach could contribute to research waste. I would recommend that each group wishing to implement a standardised set of registry items should consider existing sets first, to assess their generalisability to the setting at hand, and critically appraise the methods used. If a new set is still needed for the new setting, then a consensus process should be followed.\n\nTypos\n\n‘core outcome’ not ‘core-outcome’\n\nAbbreviations - COMET (Core Outcome Measures In Effectiveness Trials)\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "6451", "date": "22 Mar 2021", "name": "Peter Cartledge", "role": "Author Response", "response": "This article presents the items comprising a minimum data set for a registry of congenital abnormalities in Rwanda, determined through a survey of health professionals. It addresses an important topic, since the set up and maintenance of a registry is no small feat, and increasing the likelihood that key, relevant items are measured is critical. I believe the authors are proposing a ‘minimum data set’ (MDS) rather than a core outcome set (COS). A MDS typically includes a COS plus additional variables (for example baseline, risk factors and treatment variables). Use of the term ‘items’ rather than ‘outcomes’, and ‘MDS’ rather than ‘COS’, throughout would be preferable therefore. Response: Thank you for this feedback. We did find it difficult during the initial write-up to know which terminology would best describe the process. We have therefore changed the terminology in line with the reviewers feedback. Background: How did the 15 African studies reviewed determine items for their registries? This information would be of interest to the reader. Is it clear that they did not use a consensus process, and if not, how did they determine what to measure? Response: There was little or no description of how they chose the items. There was also a lack of consistency in the items that they chose. Together these two factors supported our own feeling that there was a need to develop this minimum data set. We have added some text to the discussion regarding the lack of description of how the previous studies identified their items. Methods: In round one, a long list of potential items has been created. This is not usually referred to as a ‘draft COS’ however since it consists of a list of any outcome ever measured by one or more groups rather than being based on any consensus process. Response: We didn’t include all outcomes/items. Rather, we only included those that met a pre-defined threshold of consensus for this round, which is described in the methodology as “Consensus was predefined to include any domain or item found in two or more of the identified registries or journal articles. These domains and items were then added to the first draft of the MDS.” Therefore, for this point we haven’t made any change to the manuscript. The list of items is long. Was there any randomization to address the potential for survey fatigue? Could the authors examine the data for this problem? If not, this should be discussed as a potential limitation. Response: We did not use randomization, and we certainly agree that this is a concern. We have added the following text to the limitations section of the manuscript. “The number of items found in Round-2 and -3 was long. Randomisation was not undertaken to avoid questionnaire fatigue. The items were sub-categorized into domains in an attempt to minimize this. We have not looked at the data to assess or measure for this questionnaire fatigue.” Pre-defining various elements of the process can reduce the potential for bias. Although no study protocol has been published in a journal, might there be one available online? Is there a Research Ethics Committee (REC) application where the design is described or did the authors determine that this work did not require REC approval? If the latter, a statement to this effect, with explanation, should be included. Response: In the section “Ethical issues” we have added the text “The project was undertaken as the Masters dissertation for AM at the University of Rwanda”. It already states that “This research proposal was therefore approved by the Institutional Review Board (IRB) at the University of Rwanda (155/CMHS IRB/2017).” We have therefore also added the text “The proposal was not published online or in a peer-reviewed journal.” It does not appear that an individual participant had the opportunity to review scores from other participants, to reflect on their own view, and then to rescore. The study design appears to be a series of two surveys therefore rather than a Delphi survey. This should be clarified. Response: In round-3 “Feedback was given to participants by giving the frequency the item was described in Round 1 (as a percentage) or if it was a new addition from Round 2.”. We did not however give participants the opportunity to reflect on their own-score and rescore. Results: The authors state there was an ineligible participant but continue to include their results. This should be clarified and explained, and the tables amended as appropriate. Response: We only included the excluded participant into the demographics table, not in the remaining analysis. We wanted to give an understanding of everyone who responded to the invite to take part (response-rate), but agree this is confusing. We have removed the excluded participant from the demographics table, this has no impact on the rest of the data presented. The addition of 32 items is high. What might this imply? How many of these additional items were in the final 103? Response: We feel that this implies that the initial studies/item sets from round-1 were not consistent and missed important items. We feel this supports the work that we have done to robustly identify our dataset. The number of new outcomes suggested in round 2 in the ‘syndrome/diagnosis’ domain is 6 in Table 1 but 8 in Table 3. Please clarify this inconsistency. Response: Yes, this is an error. Thank you for spotting it. This has been one of the challenges of having no funding and therefore having no Delphi-specific software and relying on excel and manual data-analysis. We have gone back to our data. There were 8 items in round 1 and 8 in round 2. We have therefore corrected the table, figure 1 and body of the text accordingly In addition to this we realize that the domains in Table 3 were in a different order to that of Table 1, we have therefore amended this to make it easier for the reader (see below) Table 2 – please add footnotes to describe the ‘Other’ categories. Response: We have added these and amended the table accordingly. Table 3 – please order ‘Patient (infant) details’ first to match Table 1. Response: Yes, we noticed this when reviewing table 1. We have amended it accordingly. There were 8 items common to the previous registries. Were these 8 in the final set? This would be an interesting discussion point. Response: We are assuming that this is in reference to the statement “with only 8 of the 283 items (3%) being present in ten or more of the 29 articles/repositories (Additional Supporting file 2).” Yes, all of these items were included in the final data-set, we have included this into the discussion. Discussion: When referring to the number of ‘outcomes’ in previous registries, is it outcomes or rather items? Response: Yes, it is items. We have amended this throughout the text. The COS-STAD standards require patients (here parents) to be involved in the determination of important outcomes. Some discussion as to why parents were not involved in this project would be of interest to the reader. Response: We had previously included a comment about this in the limitations. But we have also added this comment about COS-STAD standards. MDS and COS projects often include an in-person meeting to finalise the set. Some discussion as to whether this was considered and, if so, why it was not pursued would be of interest to the reader. Response: We have added this into the limitations, along with the following statement “This was not considered in our study. We used remote digital methods to ensure a wide range of participants without excluding the voice and opinions of anyone who would be unable to travel. A face-to-face meeting was also beyond the means of the study which was the Masters project of AM and had no funding available” We have also changed the title to “e-Delphi” to make this more clear from the start. Why is ‘the use of non-African repository data-sets in the first draft’ necessarily a limitation? Response: Maybe because the needs of a data-set in a developed nation may be different to those in this setting. Therefore, including them may have biased the early consensus. Conclusion: The authors conclude that a new set of registry items should be developed for each setting. I believe this approach could contribute to research waste. I would recommend that each group wishing to implement a standardised set of registry items should consider existing sets first, to assess their generalisability to the setting at hand, and critically appraise the methods used. If a new set is still needed for the new setting, then a consensus process should be followed. Response: Sorry, the word “develop” was poorly chosen. We have reworded this section to give a better explanation of the point we were trying to get across. Typos: ‘core outcome’ not ‘core-outcome’ Response: Amended. Abbreviations - COMET (Core Outcome Measures In Effectiveness Trials) Response: Amended." } ] }, { "id": "73403", "date": "22 Oct 2020", "name": "Bassel H. Al Wattar", "expertise": [ "Reviewer Expertise Academic obstetrician and gynecologist with experience in randomised clinical trials and core outcome sets" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for asking me to review this article describing efforts to develop a ''core outcome set'' for congenital abnormalities registry in Rwanda.\nAt its face value, I think the authors are inaccurate in calling this a core outcome set, it is rather an adoption of published outcomes in other national and international registries to what is applicable in Rwanda. In other words, this is data dictionary or minimally agreed dateset for a prospective national registry.\n\nSuch a step is certainly welcomed if a national registry is in planning to increase its value and impact on patient care. Still the key elements are missing from this exercise especially the involvement of multi-stakeholder including patient representative, health system researchers and policymakers. All of whom were absent in this Delphi.\n\nI have no major comment on the conduct of the Delphi survey, but ultimately the impact of this dataset is only relevant to Rwanda with no generalizability to the medical literature.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [ { "c_id": "6452", "date": "22 Mar 2021", "name": "Peter Cartledge", "role": "Author Response", "response": "At its face value, I think the authors are inaccurate in calling this a core outcome set, it is rather an adoption of published outcomes in other national and international registries to what is applicable in Rwanda. In other words, this is data dictionary or minimally agreed dateset for a prospective national registry. Response: Similar to reviewer 1, we have amended the terminology. Though we feel we have done more than just describing other registries. Such a step is certainly welcomed if a national registry is in planning to increase its value and impact on patient care. Still the key elements are missing from this exercise especially the involvement of multi-stakeholder including patient representative, health system researchers and policymakers. All of whom were absent in this Delphi. Response: Yes, we acknowledge they were missing from the study." } ] } ]	1	https://f1000research.com/articles/8-1037
https://f1000research.com/articles/10-13/v1	11 Jan 21	{ "type": "Case Report", "title": "Case Report: Successful revascularization in massive pulmonary embolism with a large protruding thrombus and dilated cardiomyopathy", "authors": [ "Hendri Susilo", "Rerdin Julario", "Citrawati Dyah Kencono Wungu", "Hendri Susilo", "Rerdin Julario" ], "abstract": "Pulmonary embolism is a potentially life-threatening condition. Despite advances in diagnostics, lack of consensus and delays in determining the diagnosis of pulmonary embolism are still important problems. We report the diagnosis and management of a 37-year-old man suffering from massive pulmonary embolism, a large protruding thrombus, and dilated cardiomyopathy. Echocardiography showed dilatation of all cardiac chambers, a large protruding thrombus in the right atrium to the inferior vena cava, impaired left and right ventricular systolic function, and global hypokinetic of the left ventricle with eccentric left ventricular hypertrophy. A thoracic computerized tomography scan showed pulmonary embolism with infarction. The patient’s blood pressure was 60/40 mmHg and heart rate was 110 bpm. The patient was diagnosed with high-risk acute pulmonary embolism. We gave him hemodynamic support and reperfusion therapy with a loading dose of 250,000 units of Streptokinase followed by 100,000 units/hour for 24 hours. After revascularization, the patient's hemodynamic condition improved. The diagnosis of acute pulmonary embolism is based on clinical symptoms, hemodynamic changes, or radiological examination. Unstable hemodynamic underlies high-risk stratification. Hypotension or shock results from obstruction of the pulmonary artery which causes increased right ventricular afterload and acute right ventricular dysfunction. Reperfusion with thrombolysis therapy could provide good outcomes in this patient. Prolonged anticoagulation should be given to prevent the recurrence of venous thromboembolism.", "keywords": [ "Massive pulmonary embolism", "large protruding thrombus", "unstable hemodynamic", "reperfusion" ], "content": "Introduction\n\nVenous thromboembolism, a clinical presentation of deep vein thrombosis (DVT) or pulmonary embolism, is the third most commonly found acute cardiovascular syndrome after myocardial infarction and stroke1. Pulmonary embolism is a potentially life-threatening condition. Most patients die from pulmonary embolism within the first few hours of the event. Despite advances in diagnostics, delay in determining pulmonary embolism diagnosis is still a significant problem2.\n\nPulmonary embolism contributes to approximately 300,000 deaths per year in the United States3. This makes pulmonary embolism one of the high-rank causes of cardiovascular death. In six European countries with a total population of 454.4 million, more than 370,000 deaths were related to venous thromboembolism in 2004. Of these patients, 34% died suddenly or within a few hours of the acute event, before therapy could be administered4. Clinicians should be better able to recognize the signs and symptoms of acute pulmonary embolism; thus, diagnosis and management can be determined quickly and accurately to reduce patient mortality. This case report presents successful revascularization of a massive pulmonary embolism with large intracardiac thrombus and dilated cardiomyopathy.\n\n\nCase presentation\n\nA 37-year-old Indonesian man was referred to the emergency department of Dr. Soetomo General Hospital, Indonesia, in June 2019, with complaints of shortness of breath and swollen legs. His occupation was a farmer. Two months earlier, the patient went to the public health center for a prolonged cough (± 5 months), which was sometimes accompanied by shortness of breath. In the public health center, acid-fast bacillus (AFB) testing was performed with a negative result. Based on a chest radiograph, the doctor decided to give group 1 anti tuberculosis drugs through the public health center. The treatment prescribed was fixed dose combination, 4 tablets daily taken orally, with composition of each tablet as follows: Rifampicin 150 mg, Isoniazid 75 mg, Pirazinamide 400 mg, and Ethambutol HCl 75 mg. Thus, in the emergency department, he was referred to the pulmonology department with a diagnosis of pulmonary tuberculosis.\n\nIn addition, the patient also had a history of deep vein thrombosis (DVT) of the left inferior limb; the patient had had a thrombectomy a month before his referral to the hospital. The complaints of swollen leg were slightly reduced at that time; however, had recurred again, accompanied by swelling on his right limb.\n\nThe patient had been diagnosed with diabetes mellitus and heart disease one month before admission. He received subcutaneous injection of 8 units insulin aspart three times a day before meals, captopril 6.25 mg every eight hours orally, spironolactone 25 mg once daily orally, digoxin 0.25 mg once daily orally, codeine 10mg every eight hours orally, and rivaroxaban 15 mg every twelve hours orally.\n\nWhen admited to the pulmonary ward, the patient complained of shortness of breath, accompanied by pain and swelling in both legs. His blood pressure was 120/80 mmHg, pulse 128 bpm, respiratory rate 26/minute, and SpO2 99% with 3 lpm nasal cannula. Physical examination revealed jugular venous distention, bilateral basal rales, hepatomegaly, and pitting edema in both lower extremities.\n\nIn laboratory findings, serum electrolytes revealed hypokalemia (K: 3.3 mmol/L; normal range 3.5–5.1 mmol/L), serum protein showed hypoalbuminemia (albumin: 3.1g/dL; normal range 3.4–5.0 g/dL), while other parameters were between normal limits. An electrocardiogram (ECG) showed sinus tachycardia rhythm 125 bpm, right-sided frontal axis, horizontal axis clockwise rotation, and slow progression of R waves at V1–V4 (Figure 1). A chest X-ray showed cardiomegaly, pulmonary congestion, and minimal bilateral pleural effusion (Figure 2). Echocardiographic examination revealed moderate mitral regurgitation (dilated mitral annulus), dilatation of all cardiac chambers (LVIDd 5.8 cm), visible thrombus in IVC to RA, decreased left and right ventricular systolic function (EF teich 35%, TAPSE 1.3 cm), and global hypokinetic of the left ventricle with eccentric LVH. The scans from a transthoracic echocardiogram (TTE) showing thrombus is shown in Figure 3 and Figure 4.\n\nA chest CT scan (Figure 5) showed right pulmonary artery embolism at ± 5.9 cm from bifurcation on the anterior side of the intermediate right bronchus; emboli on the left pulmonary artery bifurcation and the left pulmonary artery basal part; multiple right intraatrial hypodense lesions not showing contrast enhancement leading to a visualization of the right intraatrial thrombus; pulmonary infarction in the lateral-posterior segment of the base of the inferior lobe of the right lung, the lateral-posterior segment of the base of the inferior lobe of the left lung, and the anterior segment of the superior lobe of the left lung; and superior vena cava thrombus at VTH level 1-5. Figure 6 shows the protruded thrombus in the right atrium passing through the tricuspid valve. TTE also showed the position of the thrombus moving from the inferior vena cava towards the right atrium (Figure 7). The movement of the large protruding thrombus can be seen in supplementary video files 1–35–7.\n\nIn the course of the assessments, no clinical, laboratory, or radiological signs of pulmonary tuberculosis were found. Eventually, the patient was transferred to the cardiology ward with the assessment of dilated cardiomyopathy + acute decompensated heart failure + deep vein thrombosis of the right and left inferior limbs + right atrial thrombus + pulmonary embolism + type II diabetes mellitus. During three days of treatment, the patient received 20 mg of Furosemide by intravenous injection every eight hours, Spironolactone 50 mg once daily orally, Ramipril 5 mg once daily orally, low-dose Bisoprolol started at 1.25 mg once daily orally, subcutaneous injection of Enoxaparin 60 mg every twelve hours, and subcutaneous injection of 6 units insulin aspart three times daily before meals. However, in the course of this treatment in the cardiology ward, the patient suddenly complained of shortness of breath accompanied by chest pain and cold sweat. His blood pressure became 60/40 mmHg, heart rate 110 bpm, and respiratory rate 28–30/minute, thus showing hemodynamic instability and shock. Therefore, he was reassessed as having high risk acute pulmonary embolism, and the patient was transferred to the cardiovascular care unit (CVCU) for observation and reperfusion therapy.\n\nIn the CVCU, we gave the patient hemodynamic support with Norepinephrine starting at 50 nanograms/kg/minute by titration. Reperfusion was carried out by giving a loading dose of 250,000 units of Streptokinase intravenously for 30 minutes, followed by 100,000 units of Streptokinase per hour for 24 hours with intravenous continuous pump. After revascularization, the patient's hemodynamic condition improved until vasopressors/inotropic drugs could be tapered off. TTE also showed the disappearance of the large protruding thrombus (supplementary video files 4–5)8,9. After the patient’s condition was stable, he was transferred to the cardiology ward until the patient was discharged after one week of thrombolytic treatment. In his discharge, rivaroxaban was prescribed at a daily dose of 20 mg as an oral anticoagulant for at least three months.\n\nOne week after discharge, the patient made a follow-up visit at the cardiology outpatient clinic. At that time, it was found that the patient's symptoms and exercise tolerance had improved, and his shortness of breath and swollen leg were reduced. The patient’s adherence to treatment was good, and there was neither sign of minor nor major bleeding due to the use of anticoagulants. Anticoagulant therapy was continued, accompanied by therapy for heart failure according to guideline-directed medical therapy (GDMT) for heart failure with reduced ejection fraction (HFrEF).\n\n\nDiscussion\n\nPulmonary embolism is a critical medical emergency. It can lead to rapid deterioration of hemodynamic condition with high moratality rate10. Pulmonary embolism usually arises from thrombus originating from the deep venous system in the inferior limb. After heading to the lungs, a large protruding thrombus can attach to the branching of the main pulmonary artery or lobar branches, causing massive pulmonary embolism and hemodynamic disorders. This case report is unique as it showed massive pulmonary embolism due to a large protruding right-sided heart burden thrombus, which is rarely found in normal cases. This condition led to hemodynamic instability of the patient. This case also showed how immediate thrombolytic therapy could make the large thrombus disappear and provide a better outcome in such a patient. Pulmonary thromboembolism is not a basic disease in itself; instead, it is a complication of the underlying venous thrombosis11. On vascular ultrasound examination in the present case, thrombi were found in the common femoral vein as well as the right and left popliteal veins. From the echocardiographic examination, the presence of thrombi in the inferior vena cava and the right atrium was also found. The limitation of this case report was the unexplained etiology of hypercoagulability of this patient.\n\nThe classical presentations of pulmonary embolism are pleuritic chest pain, sudden onset, shortness of breath, and hypoxia. However, most patients with pulmonary embolism have no apparent symptoms. Conversely, symptoms can vary from sudden hemodynamic collapse to progressive shortness of breath. A diagnosis of pulmonary embolism should be suspected in patients with respiratory symptoms that cannot be explained by other alternative diagnoses12.\n\nOne of these hemodynamic instability manifestations indicates a high-risk acute pulmonary embolism13: (1) cardiac arrest; (2) obstructive shock; and (3) persistent hypotension. In this patient, clinical symptoms included shortness of breath, tachycardia, chest pain, coughing, signs of inferior extremity DVT, tachypnoea, bilateral basal rhonchi, neck venous distention, minimal bilateral pleural effusion, and hemodynamic disorders. This patient had an obstructive shock, therefore, he was classified into a high-risk acute pulmonary embolism.\n\nAll patients with pulmonary embolism need immediate risk stratification. Thrombolytic therapy should be given to patients with acute pulmonary embolism with clinical hypotension (systolic pressure <90 mm HG) who do not have a high risk of bleeding. Thrombolysis in these patients should not be delayed because of the potential for irreversible cardiogenic shock. Thrombolytic therapy is recommended in certain patients with acute pulmonary embolism indicating a high risk of hypotension at initial clinical presentation or after starting anticoagulants Assessing the severity of pulmonary embolism, prognosis, and bleeding risk determines whether thrombolytic therapy can be given. Thrombolytic therapy is not recommended for most patients with acute pulmonary embolism that is not associated with hypotension14. Primary reperfusion therapy - in most cases systemic thrombolysis - is the treatment of choice for patients with high-risk pulmonary embolism (with hemodynamic instability). Surgical pulmonary embolectomy or percutaneous catheter-directed treatment is an alternative reperfusion option in patients with contraindications to thrombolysis13.\n\nBefore conducting thrombolysis therapy, several absolute and relative contraindications must be considered. Absolute contraindications for thrombolysis are13: (1) history of hemorrhagic stroke or stroke of unknown cause; (2) ischemic stroke during the last six months; (3) neoplasms of the central nervous system; (4) major trauma, surgery or head injury in the past three weeks; (5) bleeding diathesis; and (6) active bleeding. While relative contraindications are: (1) transient ischemic attacks in the last six months; (2) the use of oral anticoagulants; (3) pregnancy or the first week postpartum; (4) non-compressible puncture sites; (5) traumatic resuscitation; (6) traumatic hypertension (systolic blood pressure > 180 mmHg); (7) severe/advanced liver disease; (8) infectious endocarditis; and (9) active peptic ulcer.\n\nAcute right heart failure with low cardiac output is the leading cause of death in patients with high-risk acute pulmonary embolism. Long-term anticoagulation is very important for preventing the recurrence of DVT or pulmonary embolism, as even if a patient has been administered anticoagulants, DVT and pulmonary embolism still often recur. Apixaban, dabigatran, rivaroxaban, and edoxaban are alternatives to warfarin as the prophylaxis and treatment of pulmonary embolism. Apixaban, edoxaban, and rivaroxaban inhibit factor Xa, while dabigatran is a direct inhibitor of thrombin15.\n\nAll patients with pulmonary embolism must be given anticoagulants for more than three months. The use of novel oral anticoagulants (NOAC) is considered to have a lower risk of bleeding than vitamin K antagonists. However, treatment with NOAC still has risks. Phase III clinical trials in venous thromboembolic patients with extended therapy show that major bleeding rates are around 1% and clinically relevant non-major bleeding is around 6%16,17.\n\nIn our patient, after the diagnosis of acute pulmonary embolism had been established, thrombolysis was the first choice. Reperfusion was carried out by giving a loading dose of 250,000 units of Streptokinase intraveneously for 30 minutes, then followed by 100,000 units of Streptokinase per hour for 24 hours. Hemodynamic support was performed by giving Norepinephrine from 50 nanograms/kg/minute by titration.\n\n\nConclusion\n\nWe reported the case of a 37-year-old man with massive pulmonary embolism caused by a large protruded thrombus and dilated cardiomyopathy. The diagnosis of acute pulmonary embolism was based on clinical symptoms, hemodynamic changes, echocardiographic examination, and a chest CT scan. Unstable hemodynamic conditions classified this patient in the high-risk stratification. Hypotension or shock resulted from acute right ventricular dysfunction due to the obstruction of the pulmonary artery embolus, which caused an increase in right ventricular afterload. Inotropic agents or vasopressors with Norepinephrine were needed to improve the hemodynamic profile. Successful revascularization was performed by thrombolysis with Streptokinase, which gave good outcomes in this patient. In conclusion, early diagnosis, risk assessment, and prompt treatment are important to treat patients with massive pulmonary embolism due to a large protruding thrombus. Hemodynamic deterioration, such as hypotension and shock, should be monitored in patients with massive pulmonary embolism to reduce mortality. Reperfusion therapy should be administered soon for patients with high-risk pulmonary embolism after assessing indication and contraindication for thrombolytic treatment. The problem with the current management of pulmonary embolism is when to start reperfusion therapy in patients with intermediate-high risk. Further research is needed to determine the right management and immediate prompt treatment in such patients.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patient.", "appendix": "References\n\nRaskob GE, Angchaisuksiri P, Blanco AN, et al.: Thrombosis: A Major Contributor to Global Disease Burden. Arterioscler Thromb Vasc Biol. 2014; 34(11): 2363–71. PubMed Abstract \| Publisher Full Text\n\nOzsu S, Oztuna F, Bulbul Y, et al.: The role of risk factors in delayed diagnosis of pulmonary embolism. Am J Emerg Med. 2011;. 29(1): 26–32. PubMed Abstract \| Publisher Full Text\n\nHeit JA, Cohen AT, Anderson Jr. FA, et al.: Estimated Annual Number of Incident and Recurrent, Non-Fatal and Fatal Venous Thromboembolism (VTE) Events in the US. Blood. 2005; 106(11): 910. Publisher Full Text\n\nCohen AT, Agnelli G, Anderson FA, et al.: Venous thromboembolism (VTE) in Europe. The number of VTE events and associated morbidity and mortality. Thromb Haemost. 2007; 98(4): 756–64. PubMed Abstract \| Publisher Full Text\n\nSusilo H, Julario R, Wungu C: Movement of the large protruding thrombus (1). f1000research.com. Media. 2020. http://www.doi.org/10.6084/m9.figshare.13475550.v1\n\nSusilo H, Julario R, Wungu C: Movement of the large protruding thrombus (2). f1000research.com. Media. 2020. http://www.doi.org/10.6084/m9.figshare.13475721.v1\n\nSusilo H, Julario R, Wungu C: Movement of the large protruding thrombus (3). f1000research.com. Media. 2020. http://www.doi.org/10.6084/m9.figshare.13475760.v1\n\nSusilo H, Julario R, Wungu C: Disappearance of large protruding thrombus after revascularization (4). f1000research.com. Media. 2020. http://www.doi.org/10.6084/m9.figshare.13475799.v1\n\nSusilo H, Julario R, Wungu C: Disappearance of large protruding thrombus after revascularization (5). f1000research.com. Media. 2020. http://www.doi.org/10.6084/m9.figshare.13475844.v1\n\nCharif F, Mansour MJ, Hamdan R, et al.: Free-Floating Right Heart Thrombus with Acute Massive Pulmonary Embolism: A Case Report and Review of the Literature. J Cardiovasc Echogr. 2018; 28(2): 146–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGoldhaber SZ, Bounameaux H: Pulmonary embolism and deep vein thrombosis. Lancet. 2012; 379(9828): 1835-46. PubMed Abstract \| Publisher Full Text\n\nWorsley DF, Alavi A: Comprehensive analysis of the results of the PIOPED Study. Prospective Investigation of Pulmonary Embolism Diagnosis Study. J Nucl Med. 1995; 36(12): 2380–7. PubMed Abstract\n\nKonstantinides SV, Meyer G, Becattini C, et al.: 2019 ESC Guidelines for the diagnosis and management of acute pulmonary embolism developed in collaboration with the European Respiratory Society (ERS). Eur Heart J. 2020; 41(4): 543–603. PubMed Abstract \| Publisher Full Text\n\nGuyatt GH, Akl EA, Crowther M, et al.: Executive summary: Antithrombotic Therapy and Prevention of Thrombosis, 9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines. Chest. 2012; 141(2 Suppl): 7S–47S. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBüller HR, Prins MH, Lensing AWA, et al.: Oral rivaroxaban for the treatment of symptomatic pulmonary embolism. N Engl J Med. 2012; 366(14): 1287–97. PubMed Abstract \| Publisher Full Text\n\nHelmert S, Marten S, Mizera H, et al.: Effectiveness and safety of apixaban therapy in daily-care patients with atrial fibrillation: results from the Dresden NOAC Registry. J Thromb Thrombolysis. 2017; 44(2): 169–178. PubMed Abstract \| Publisher Full Text\n\nBeyer-Westendorf J, Förster K, Pannach S, et al.: Rates, management, and outcome of rivaroxaban bleeding in daily care: Results from the Dresden NOAC registry. Blood. 2014; 124(6): 955–62. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "77083", "date": "12 Jan 2021", "name": "Henry Sutanto", "expertise": [ "Reviewer Expertise Cardiovascular disease", "cardiogenetics", "cardiac arrhythmias", "arrhythmogenesis", "inherited arrhythmia syndromes", "computational modeling and bioinformatics." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this case report, Dr. Susilo and colleagues reported a case of a man with pulmonary embolism (PE), right atrial (RA) thrombus and dilated cardiomyopathy (DCM). In general, the manuscript showed nicely the rapidly progressing PE into hemodynamic instability, which was immediately corrected following the administration of thrombolytic agent. However, I have some major and minor comments to be addressed by the authors, including the completeness of data.\nOverall, the authors need to highlight the distinctiveness of this case, which is not well-defined in the current version of the manuscript. It is currently unclear why this case is different than the other PE cases? The fact that one of the thrombi is located in the RA does help to stress the importance of this case report.\n\nI would suggest the authors to arrange and discuss the data more systematically, and provide complete history and supporting data in the manuscript. For example, the patient was diagnosed with diabetes mellitus and heart disease 1-month before admission. Which heart disease? What about the glycemic status prior to and after admission? Was the diabetes controllable?\n\nThe time course of clinical and laboratory parameters is lacking. For example, it is currently unclear about the ejection fraction after discharge and during the follow-up visit? What about the hypercoagulable state and so on? These values are important to report in this manuscript.\n\nHere, the authors explicitly mentioned three observed phenomena in the title: 1) PE; 2) RA thrombus and 3) DCM. However, the manuscript mainly focused on the PE and RA thrombus with a very limited discussion regarding DCM and its correlation with the other observed pathologies. Please clarify the reason and adapt the discussion accordingly elaborating the potential interactions between those pathologies.\n\nIn addition to the clinical presentations, please include the ECG recordings and other data justifying the diagnosis of acute PE during the shock event.\n\nThe authors are requested to speculate on the origin of the RA thrombus. Is it primarily from the RA or is it a migrating thrombus? Was there any evidence of situations facilitating thrombus formation, such as hypercoagulability, atrial fibrillation, tricuspid valve disease, and so on prior to the incident? Provide some supporting data (e.g., lab results, etc.).\n\nThe authors mentioned that the etiology of hypercoagulability in this patient is unknown. However, laboratory results showing hypercoagulability were not reported. Please add them into the study. What about the link between diabetes and hypercoagulable state, which has been discussed by many other publications? Please discuss this.\n\nWas left ventricular (LV) noncompaction observed in this patient? Elaborate your arguments.\n\nThe rationale of DCM diagnosis in this patient is unclear. The authors need to provide a table containing checklist of clinical, imaging, and genetic data supporting this diagnosis. The authors can benefit from the paper by Mestroni et al. (Eur Heart J. 1999)1 or Mathew et al. (Echo Res Pract. 2017)2.\n\nPlease confirm the nature of DCM in this patient? Familial or acquired? Include the family pedigree if there is an evidence of inherited DCM.\n\nPatients with DCM have a high risk of sudden cardiac death. Please explain the reason why in this patient, implantable cardioverter defibrillator (ICD) was not (planned to be) implanted? Moreover, the patient was hypokalemic, which poses to a bigger risk for life-threatening cardiac arrhythmias. Please also clarify the approach has been done to fix this problem.\n\nThe authors mentioned in the abstract that echocardiography showed dilation of all cardiac chambers. However, no quantitative assessment of the atrial diameter provided. Please add.\n\nI am still not sure why anti-tuberculosis drugs were prescribed while AFB test was negative? Is there any involvement of these drugs on the clinical presentation of the patient?\n\nWhat is the rationale of administering rivaroxaban in the patient prior to the hospital admission? Was that due to the DVT? Please speculate on the reason why thrombi still persisted after rivaroxaban therapy (perhaps could also be supported by patient's lab results)?\n\nIn Figure 5, please add some details / labels on the abnormalities observed in the CT scan, including the location of thrombi in the pulmonary artery.\n\nAvoid using numbers 1), 2), 3) in the text. Convert them into words: first, second, etc. if necessary.\n\nIn the abstract: “We gave him …” please change into passive sentence similar to the rest of the manuscript.\n\nIn the abstract: please add the administration route of streptokinase.\n\nPlease convert the incidence of PE in six EU countries in the introduction into 1 in XXX. The way the authors reported the epidemiological data is not common.\n\n“be better able to” should be “be able to better …”\n\n“… determined quickly and accurately to reduce patients’ mortality”\n\n“the patient had undergone a thrombectomy a month before …”\n\n“SpO2 99% with 3 lpm nasal cannula” should be “3 liter per minutes oxygen via nasal cannula”.\n\n“moratality” should be “mortality”\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [ { "c_id": "6309", "date": "15 Feb 2021", "name": "Citrawati Wungu", "role": "Author Response", "response": "Thank you for your review. We have made revision according to your suggestions. These are our explanations regarding the comments: We have added the distinctiveness of this case, that this was a rare case of protruded thrombus in the right atrium passing through the tricuspid valve which position moving from the inferior vena cava towards the right atrium, causing a massive pulmonary embolism and dilated cardiomyopathy. We have added the history and supporting data as suggested. We have added the explanation of the ejection fraction which had not improved at the follow-up visit. Ejection fraction was not an indicator for discharge in such patient. We have stated in the last paragraph of discussion section that the limitation of this case report was the unexplained etiology of hypercoagulability of this patient. In this case report we did not focus on the DCM. However, DCM increases the risk of intracardiac thrombus which causes hypokinetic condition (venous stasis), a part of Virchow’s triad. ECG in PE is less specific. The most common ECG finding in the setting of a pulmonary embolism is sinus tachycardia. S1Q3T3 is a specific pattern in PE, however, this pattern only occurs in about 10% of people with pulmonary embolisms and is similar to the ECG findings of a left posterior fascicular block, or LPFB (Mohammed and Elsayed, 2020). It was a migrating thrombus from proximal femoral vein. The patient had a history of proximal bilateral femoral deep vein thrombosis (DVT) of the left inferior limb proven by ultrasonography examination and the patient had had a thrombectomy a month before his referral to the hospital. The complaints of swollen leg were slightly reduced at that time; however, had recurred again, accompanied by swelling on his right limb. He had no atrial fibrillation or tricuspid valve disease. Endothelial abnormalities undoubtedly play a role in the enhanced activation of platelets and clotting factors seen in diabetes. Coagulation activation markers, such as prothrombin activation fragment 1+2 and thrombin-anti-thrombin complexes, are elevated in diabetes. The plasma levels of many clotting factors including fibrinogen, factor VII, factor VIII, factor XI, factor XII, kallikrein, and von Willebrand factor are elevated in diabetes. Conversely, the level of the anticoagulant protein C (PC) is decreased. The fibrinolytic system, the primary means of removing clots, is relatively inhibited in diabetes due to abnormal clot structures that are more resistant to degradation and an increase in plasminogen activator inhibitor type 1 (PAI-1). Increased circulating platelet aggregates, increased platelet aggregation in response to platelet agonists, increased platelet contractile force (PCF), and the presence of higher plasma levels of platelet release products, such as beta-thromboglobulin, platelet factor 4, and thromboxane B(2), demonstrate platelet hyperactivity in diabetes (Carr, 2001). However, our opinion is that the extensive thrombosis condition in this patient was not solely due to his diabetes.Hypercoagulability conditions can be related to genetic disorders, for example: Factor V Leiden (the most common), Prothrombin gene mutation, deficiencies of natural proteins that prevent clotting (such as antithrombin, protein C and protein S), elevated levels of homocysteine, elevated levels of fibrinogen or dysfunctional fibrinogen (dysfibrinogenemia), elevated levels of factor VIII (still being investigated as an inherited condition) and other factors including factor IX and XI, abnormal fibrinolytic system, including hypoplasminogenemia, dysplasminogenemia, and elevation in levels of plasminogen activator inhibitor (PAI-1) (Khan and Dickerman, 2006; Jan, 2020). However, we acknowledge that the weakness of this case report was that we did not have data for any genetic abnormalities. There were no signs of LV noncompaction in this patient. No family history data was obtained. The Echo result did not show any trabeculations. No genetic data available. DCM was diagnosed based on clinical and structural abnormalities by echocardiography. We found large ventricular enlargement with global hypokinetic and reduced ejection fraction. ICD is indicated in heart failure patients with reduced EF (EF < 35) and no improvements with optimal medical treatment based on GDMT (Ponikowski et al., 2016). In this patient we found high burden intracardiac thrombus, thus it will be so dangerous for us to perform ICD. It can make the thrombus escape dan migrate, thus we focus on the emergency and gave GDMT as the optimal treatment. We have added atrial diameter as follows: LA mayor 6.4 cm, LA minor 47 cm; RA mayor 5.5 cm, RA minor 4.5 cm, RVDB 3.2 cm. In the public health center, the doctor diagnosed the patient with clinical TB with negative AFB, however, in the course of the assessments in the hospital, no clinical, laboratory, or radiological signs of pulmonary tuberculosis were found, thus the patient was transferred to the cardiology ward. Rivaroxaban was prescribed as a DVT therapy that has been known before by the referring doctor. Persistent thrombus may be due to the patient's hypercoagulability condition. However, we cannot give more specific conclusions regarding this matter due to limited data and laboratory examination results. We have inserted the arrow to the figure. In manuscript we have stated that the CT scan showed right pulmonary artery embolism at ± 5.9 cm from bifurcation on the anterior side of the intermediate right bronchus. We have made changes to the formatting as suggested. References: Carr, M. E. (2001) ‘Diabetes mellitus: a hypercoagulable state.’, Journal of diabetes and its complications, 15(1), pp. 44–54. doi: 10.1016/s1056-8727(00)00132-x. Jan, B. S. P. T. A. G. A. (2020) Hypercoagulability. Treasure Island (FL): StatPearls Publishing. Available at: https://www.ncbi.nlm.nih.gov/books/NBK538251/. Khan, S. and Dickerman, J. D. (2006) ‘Hereditary thrombophilia’, Thrombosis Journal, 4, pp. 1–17. doi: 10.1186/1477-9560-4-15. Mohammed, Y. and Elsayed, H. (2020) ‘McGinn-White Sign or S1Q3T3-Pattern in Pulmonary Embolism ; Significance and Differential Diagnosis ; Narrative Updating Review’, International Journal of Research Studies in Medical and Health Sciences, 5(11), pp. 15–24. Available at: http://www.ijrsmhs.com/pdf/v5-i11/2.pdf. Ponikowski, P. et al. (2016) ‘2016 ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure’, European Heart Journal, 37(27), pp. 2129-2200m. doi: 10.1093/eurheartj/ehw128." } ] } ]	1	https://f1000research.com/articles/10-13
https://f1000research.com/articles/10-77/v1	05 Feb 21	{ "type": "Research Article", "title": "Population characterization and parasitological assessment of the giant African snail (Achatina fulica) in urban areas of Cartagena, Colombia", "authors": [ "Eder Cano-Pérez", "Jaison Torres-Pacheco", "Luis Barraza-Quiroz", "Jorge Morelos-Muñoz", "Doris Gómez-Camargo", "Eder Cano-Pérez", "Jaison Torres-Pacheco", "Luis Barraza-Quiroz", "Jorge Morelos-Muñoz" ], "abstract": "Background: The giant African snail, Achatina fulica, is an invasive species recognized for being a serious agricultural pest and an intermediary vector for diverse parasites that cause diseases in humans. The knowledge of the state of African snail populations in urban areas is of great ecological and public health importance. Therefore, our objective was to characterize the status of giant African snail populations present in the city of Cartagena, Colombia, including the assessment of nematode parasites in the specimens. Methods. Sites were visited following information from citizens affected by the presence of the African snail. The specimens were collected and transported to the laboratory; subsequently, they were weighed, measured, and classified by size. Dissections of lung tissue and soft organs were performed to search for and identify nematode parasites. Size measurement between the sampled sites was statistically compared and density and biomass indicators were established. Results. In total, 204 snails were collected distributed among four sites within Cartagena city. Of these, 50% were juvenile specimens (10-40 mm). The size of the specimens showed significant differences between the sampling areas. The calculated density was between 0.0019-0.68 ind/m2 and the biomass between 3.92-48.75 kg/ha. No presence of nematode parasites was observed in these specimens. Conclusions. Densities and biomasses of A. fulica in Cartagena do not reach levels considered highly harmful. On the other hand, although no parasites were found in these snails, it is relevant to continue with studies on the human health risks that represent the presence of this invasive species in urban areas of Cartagena.", "keywords": [ "Achatina fulica", "biomass", "population density", "invasive species", "plague", "intermediary vector" ], "content": "Introduction\n\nOne of the greatest threats to global biodiversity, livelihoods, human and animal health, agriculture, and the local economy are invasive alien species1. The giant African snail, Achatina fulica, (Bowdich 1822) is a gastropod native to Africa and considered one of the 100 worst invasive alien species in the world2,3. Currently, this species is widespread in tropical and subtropical areas on all continents due to the growth of international trade and increased transport that facilitate the introduction of this species, accidentally through agricultural or horticultural products or intentionally as a source of food or as a pet2,4,5.\n\nThe giant African snail is recognized as an agricultural and garden pest5. Its great reproductive capacity allows it to reach high densities and biomass in short times3,5, and its broad spectrum herbivorous diet allows it to consume more than 50 species of native and agricultural plants, all of which makes this species a serious pest with the ability to modify habitats and displace native species5. The giant African snail is an intermediate host for various parasites, among which the nematodes Angiostrongylus cantonensis and Angiostrongylus costaricensis stand out, which cause eosinophilic meningoencephalitis and abdominal angiostrongyliasis in humans, respectively6,7. Recently, the presence of A. cantonensis in African snails in Colombian territory was confirmed, putting the population on alert due to the exposure of humans to the parasitic load of this snail, leading to the consideration of eosinophilic meningitis as a possible emerging disease in Colombia8. Risk factors for infection in humans and pets by this and other nematodes include eating infected snails raw or undercooked, or food contaminated by snail slime or feces9,10.\n\nIn Colombia, the presence of A. fulica was registered for the first time in 2010, since then the mollusk has been distributed in more than 20 departments, registering in all regions of the country11. Particularly in the Colombian Caribbean Region, the growth of A. fulica populations has been documented in the department of Sucre11–13. In Cartagena, Bolívar, journalistic media reported the presence of the giant African snail south of the city in 2016. However, since its appearance, there has been no scientifically documented record of the presence and distribution of this species in urban areas of the city, as well as its impact on human health. Therefore, the objective of this study was to generate the baseline of reference information on the African snail populations present in the Cartagena city, including the assessment of nematode parasites in the specimens.\n\n\nMethods\n\nCartagena (10 ° 23'59 '' N 75 ° 30'52 '' W), capital of the department of Bolívar located within the Colombian Caribbean region, is a city of low ecotope or coastal, presenting altitudes between 1 and 9 meters above sea level, a temperature average of 27.5°C, a relative humidity of 80% and an annual rainfall of 870 mm.\n\nThe search for, and collection of, the snails was carried out between July and October 2019, a period that included the rainy season in the region and which the proliferation of the giant African snail is favored. The sampling consisted of visiting the sites within the city of Cartagena previously informed by citizens affected by the presence of the snail in their homes and surroundings. During the inspections, searches were carried out in gardens, under undergrowth and rubble, or between crevices where they could take refuge. The specimens were collected manually using gloves and face masks and transported in easy-sealing bags to the laboratory of the UNIMOL research group in the University of Cartagena. As this was a monitoring study, all the snails available in the study period were included for analyzes, no exclusion criteria were applied for the collection of the snails.\n\nThe collection included live individuals of all sizes, which were measured using a digital caliper (0.01 mm) and weighed in a weighing machine (1 gr). The length of the shell was used as a descriptor of the total size of each individual, so size classes were defined as the following: class 1, to which newly hatched individuals and up to 10 mm were assigned; class 2, juveniles (10–40 mm); class 3, young adults (40–70 mm) and class 4, adults (> 70 mm)14.\n\nSpecimens larger than 50 mm have a greater probability of carrying nematode larvae15, even so, we decided to evaluate specimens greater than 30 mm to increase the probability of findings. The snails were washed externally and were put to sleep by thermal shock by introducing them in ice water, once they were numb, the shell was broken by using dissection scissors to extract the lung tissue and the rest of the soft organs. The search and collection methods for nematode parasites described by Córdoba et al.16 were used. Briefly, the lung tissue was cut in 1 × 1 cm pieces and placed on microscope slides for observation under a stereoscope. Likewise, the other organs were cut and examined by compression between two glass pieces.\n\nAs data were not normally distributed, the snail size measurements between the sites were compared using the Kruskal-Wallis and Bonferroni tests post hoc, using the statistical software SPSS v.19.0 (IBM Corp, Armonk, NY) with a statistic significance defined as p<0.05. Also, the population density of each zone was calculated according to the number of individuals collected per effective area sampled (Individuals/m2) and the biomass per unit area (kg/hectares) was established.\n\nGiant African snail has been declared in Colombia as an invasive species, for which legal research permits are not required. This study was adjusted to the management regulations for A. fulica issued by the Ministry of Environment, 201117. Also, during the execution of the study, efforts were made to reduce animal suffering during sacrifices, for this, the snails were put to sleep in ice water until their death before the dissections.\n\n\nResults\n\nDuring the period studied 204 snails were collected, distributed in four sites in Cartagena for which the presence of A. fulica was indicated by the community (Figure 1). The inspected sites where the snails were collected corresponded essentially to the gardens of private properties and residential condominiums (Manga, Las Gavias, and Serena del Mar). At these sites the snails were found in different substrates, such as plants, tree roots, grasses, under litter, flowerpots and ornamental plants; In Zaragocilla, aggregates were found in the rubble.\n\nOverall, 50% of the total material collected corresponded to class 2 snails, followed by class 3 (42.64%) and class 4 (7.35%). In particular, in the Zaragocilla and Las Gavias sites, class 3 individuals predominated with 44.11% and 52.33%, respectively, in Manga, class 2 specimens were more abundant with 73.33% (Table 1).\n\nClass 1: newly hatched individuals (up to 10 mm); class 2: juveniles (10 to 40 mm); class 3: young adults (40 to 70 mm); class 4: adults (> 70 mm).\n\nMean size and weight per site is shown in Table 2. The Kruskal-Wallis test showed a significant difference between the sizes of the most abundant African snail population groups (Zaragocilla, Manga, and Las Gavias sites) (p<0.01). Likewise, Bonferroni-adjusted pairwise comparisons indicated that differences existed between each site (p<0.01 between each pairing). The population density established in each point, as well as the estimated biomass according to the calculated total weight, is presented in Table 3.\n\nn: number of snails collected, SD: standard deviation.\n\nn: number of snails collected.\n\nRegarding the parasitological analyzes, all the observed lungs were found healthy (free of cysts), likewise, no free larvae were observed in the slime or the rest of the soft organs, showing that these specimens were not infected with nematode parasites according to the methodology used.\n\n\nDiscussion\n\nUnderstanding the populations of giant African snail present in different areas of Colombia is of great ecological, agricultural, and public health importance. The current study constitutes the first scientifically documented record of the presence of the species A. fulica in urban areas of the city of Cartagena.\n\nIt is known that the African snail can reach sizes of up to 300 mm in the length of the shell18. In contrast, other studies establish that the size naturally reached by A. fulica is around 100 mm19,20. Even so, the mean values reported are usually much smaller and vary between 30 and 60 mm12,13,21–24. Comparatively, our results from Cartagena agree with these last values, obtaining average sizes between 36.2–69 mm. Patterns in snail size can reflect the age structure of the population in a given area. It has been proposed that the establishment of A. fulica in new areas takes place in three stages: exponential, known as a long phase with vigorous individuals; stable, with a prevalence of a variable shell size among young and adult, and the decline phase, where young individuals are prevalent21. This suggests that the populations obtained from A. fulica collected in Cartagena are in the stable phase, presenting a variety of young and adult individuals.\n\nThe population density of A. fulica reported in this study was between 0.0019–0.6818 ind/m2, these density values are similar to those obtained by De la Ossa et al. in studies carried out with snails collected in urban areas of the city of Sincelejo and other municipalities of the department of Sucre, these authors reported densities in a range of 0.0031–0.205 ind/m212,13,23. Other similar values were reported in a study performed in Ilha Porchat, Brazil, obtaining a density of 0.07 ind/m23. Investigations such as those carried out in Havana, Cuba, showed a considerably lower density of snails (0.00015 ind/m2)25. In contrast, densities of 1.1–4.6 ind/m2 have been reported in different departments of Colombia26, 0.06–8 ind/m2 in Northeast Brazil27, and an average of 8.4 ind/m2 in Puyo, Ecuador21. On the other hand, studies performed in Puerto Iguazú y Corrientes, Argentina, recorded a much higher average density, which reaches 107.6 ind/m2 and 118.6 ind/m2, respectively14,28. It has been mentioned that the areas highly affected by the giant African snail present densities of 10 ind/m2 or more11,29; this similarly occurs with regards to biomass, where devastating values of up to 780 kg/ha are estimated for areas which are highly affected11,30. If that is the case, the densities and biomasses obtained in the snail populations of Cartagena are not very harmful.\n\nNematodes such as A. cantonensis and A. costaricensis cause eosinophilic meningoencephalitis and abdominal angiostrongyliasis, respectively, in humans. The life cycle of these nematodes involves rodents as the definitive host and mollusks of various species, including A. fulica, as intermediate hosts7. The snails examined in this work did not show evidence of infection by nematode parasites, one of the reasons could be the environment in which these snails were found. It has been noted that the African snail prefers sites such as garbage dumps, landfills, and empty lots, places where there is a greater probability of contact with rats, necessary for the nematodes to complete their life cycle16. In our case, more than 80% of the analyzed snails were collected in gardens of remarkably well-kept properties given the exclusivity of the sites, and according to interviews with various neighbors, rodents do not frequent these spaces.\n\nIn conclusion, although the densities of A. fulica estimated do not reach values considered highly harmful, the presence of this invasive species in properties and residential areas of Cartagena is worrying. Most of the snails were found associated with gardens, which suggests that the introduction and dispersal of this species within the city are related to the transport of organic fertilizers or other garden products, which could convert this species into a serious pest. Similarly, although no parasites were found in these snails, it is necessary to continue carrying out studies on the effect of this species on human health, as well as training campaigns on the management and control of the giant African snail in the city.\n\n\nData availability\n\nFigshare: Raw data file_African Snail Project.csv, https://doi.org/10.6084/m9.figshare.1328880831.\n\nThis project contains the following underlying data:\n\nRaw data file_African Snail Project.csv (original raw data)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nThe authors would like to thank the citizens of Cartagena and the Environmental Public Establishment of Cartagena (EPA-Cartagena) for their timely reports that allowed the location of the sites affected by the African snail. We thank the medical student Mariangel Sandoval for her special assistance in the collection of the specimens. Finally, we thank the team of the UNIMOL research group and DADIS for their support.\n\n\nReferences\n\nSimberloff D, Martin JL, Genovesi P, et al.: Impacts of biological invasions: what's what and the way forward. Trends Ecol Evol. 2013; 28(1): 58–66. PubMed Abstract \| Publisher Full Text\n\nRekha Sarma R, Munsi M, Ananthram AN: Effect of Climate Change on Invasion Risk of Giant African Snail (Achatina fulica Férussac, 1821: Achatinidae) in India. PLoS One. 2015; 10(11): e0143724. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMiranda MS, Fontenelle JH, Pecora IL: Population structure of a native and an alien species of snail in an urban area of the Atlantic Rainforest. Journal of Natural History. 2015; 49(1–2): 19–35. Publisher Full Text\n\nSilva ECd, Omena EP: Population dynamics and reproductive biology of Achatina fulica Bowdich, 1822 (Mollusca, Gastropoda) in Salvador-Bahia. Biota Neotrop. 2014; 14(3): 1–11. Publisher Full Text\n\nRaut SK, Baker GM: Achatina fulica Bowdich and other Achatinidae as pests in tropical agriculture. In: Barker GM, editor. Molluscs as Crop Pests. Wallingford: CAB International. 2002; 55–114. Publisher Full Text\n\nLondoño JD, Zamora A, Osorio J: Angiostrongylus Cantonensis y el caracol africano gigante como causantes de meningitis eosinofilica. RFS Revista Facultad de Salud. 2013; 5(2): 61–9. Publisher Full Text\n\nMaldonado A, Simoes R, Thiengo S: Angiostrongyliasis in the Americas. In: Lorenzo-Morales J, editor. Zoonosis. Brasil: IntechOpen; 2012. Publisher Full Text\n\nGiraldo A, Garzón C, Castillo A, et al.: Confirmation of the presence of Angiostrongylus cantonensis in lung tissue of the African giant snail (Lissachatina fulica) in Colombia. Infectio. 2019; 23(2): 129–32. Publisher Full Text\n\nAlfaro-Alarcón A, Veneziano V, Galiero G, et al.: First report of a naturally patent infection of Angiostrongylus costaricensis in a dog. Vet Parasitol. 2015; 212(3–4): 431–4. PubMed Abstract \| Publisher Full Text\n\nd'Ovidio D, Nermut J, Adami C, et al.: Occurrence of Rhabditid Nematodes in the Pet Giant African Land Snails (Achatina fulica). Front Vet Sci. 2019; 6: 88. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDe la Ossa-Lacayo A, De la Ossa V, Lasso C: Registro del caracol africano gigante Achatina fulica (Bowdich 1822) (Mollusca: Gastropoda-Achatinidae) en Sincelejo, costa Caribe de Colombia. Biota Colomb. 2012; 13(2): 247–52. Reference Source\n\nDe la Ossa-Lacayo A, De la Ossa V: Caracol africano gigante Achatina fulica bowdich 1822 (Mollusca: Gastropoda-achatinidae) en zona urbana de Sincelejo y Sampués Sucre, Colombia. Rev Colombiana Cienc Anim Recia. 2014; 6(2): 299–304. Publisher Full Text\n\nDe La Ossa-Lacayo A, Castro J, Monroy M: Ampliación de la presencia del caracol africano gigante Acathina fulica (Bowdich 1822) (Mollusca: Gastropoda-Achatinidae) en la zona norte del departamento de Sucre, Colombia. Rev Colombiana Cienc Anim Recia. 2017; 9(2): 317–22. Publisher Full Text\n\nGutierrez DE, Beltramino AA, Vogler RE, et al.: Expansión del rango de distribución de Achatina fulica Bowdich, 1822 (Gastropoda) en la Argentina y su concordancia con modelos predictivos. Amici Molluscarum. 2013; 21(1): 17–21. Reference Source\n\nFranco-Acuña DO, Pinheiro J, Torres EJL, et al.: Nematode cysts and larvae found in Achatina fulica Bowdich, 1822. J Invertebr Pathol. 2009; 100(2): 106–10. PubMed Abstract \| Publisher Full Text\n\nCórdoba D, Patiño A, Giraldo A: Prevalence of Strongylida nematodes associated with African Snail, Achatina fulica, in Valle del Cauca, Colombia. Rev MVZ Córdoba. 2017; 22(3): 6276–86. Publisher Full Text\n\nResolución Número 654 de Abril 7 2011: Por el cual se corrige la Resolución No. 0848 del 23 de mayo de 2008 y se adoptan las medidas que deben seguir las autoridades ambientales, para la prevención, control y manejo de la especie Caracol Gigante Africano (Achatina fulica). Diario Oficial: n 48.041; 2011. Reference Source\n\nVinci G, Unnithan VK, Sugunan VV: Farming of the Giant African Snail, Achatina fulica. India. Central Inland Capture Fisheries Research Institute. 1998; 56: 1–24.\n\nAlbuquerque F, Peso-Aguiar M, Assunção-Albuquerque M, et al.: Do climate variables and human density affect Achatina fulica (Bowditch) (Gastropoda: Pulmonata) shell length, total weight and condition factor? Braz J Biol. 2009; 69(3): 879–85. PubMed Abstract \| Publisher Full Text\n\nRauth S, Ghara T: Impact of individual’s size on density of the snail pest Achatina fulica Bowdich (Gastropoda: Achatinidae). Boll Malacol. 1989; 25(9–12): 301–6. Reference Source\n\nGołdyn B, Kaczmarek Ł, Roszkowska M, et al.: Urban Ecology of Invasive Giant African Snail Achatina fulica (Férussac) (Gastropoda: Achatinidae) on its First Recorded Sites in the Ecuadorian Amazon. Am Malacol Bull. 2017; 35(1): 59–64. Publisher Full Text\n\nde Andrade-Porto SM, de Souza KCP, Cárdenas MQ, et al.: Occurrence of Aelurostrongylus abstrusus (Railliet, 1898) larvae (Nematoda: Metastrongylidae) infecting Achatina (Lissachatina) fulica Bowdich, 1822 (Mollusca: Gastropoda) in the Amazon region. Acta Amazon. 2012; 42(2): 245–50. Publisher Full Text\n\nDe La Ossa J, De La Ossa-Lacayo A, Castro J, et al.: Incremento poblacional de Achatina fulica Bowdich, 1822 (Mollusca: Gastropoda - Achatinidae) en una zona urbana de Sincelejo, Sucre, Colombia. Rev Asoc Col Cienc (Col). 2017; 1(29): 21–9. Reference Source\n\nPatiño A, Murillo O, Giraldo A: Variación morfológica poblacional de una especie invasora: el caracol gigante africano, Achatina fulica (Bowdich 1822) (Mollusca: Gastropoda-Achatinidae) en el departamento del Valle del Cauca, Colombia. Biota Colomb. 2018; 19(1): 112–22. Reference Source\n\nVázquez AA, Sánchez J: First record of the invasive land snail Achatina (Lissachatina) fulica (Bowdich, 1822) (Gastropoda: Achatinidae), vector of Angiostrongylus cantonensis (Nematoda: Angiostrongylidae), in Havana, Cuba. Molluscan Research. 2015; 35(2): 139–42. Publisher Full Text\n\nAvendaño JM, Linares EL: Morfometría del caracol gigante africano Achatina fulica (Gastropoda: Achatinidae) en Colombia. Cuadernos de Investigación UNED. 2015; 7(2): 287–93. Reference Source\n\nAlbuquerque F, Peso M, Assunção M: Distribution, feeding behavior and control strategies of the exotic land snail Achatina fulica (Gastropoda: Pulmonata) in the northeast of Brazil. Braz J Biol. 2008; 68(4): 837–42. PubMed Abstract \| Publisher Full Text\n\nGutiérrez Gregoric D, Núñez V, Vogler R, et al.: Invasion of the Argentinean Paranense Rainforest by the Giant African Snail Achatina fulica. Am Malacol Bull. 2011; 29(1/2): 135–7. Publisher Full Text\n\nLake PS, O’Dowd DJ: Red crabs in rainforest, Christmas Island: biotic resistance to invasion by an exotic snail. Oikos. 1991; 62(1): 25–29. Publisher Full Text\n\nTiller S: Production et cycle reproducteur de l’escargot Achatina fulica Bowdich, 1822 en Nouvelle Caledonie (Pulmonata: Stylommatophora: Achatinidae). Haliotis. 1982; 12: 112.\n\nCano Pérez E, Torres-Pacheco J, Barraza-Quiroz L, et al.: Raw data file_ African Snail Project.csv. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.13288808.v1" }	[ { "id": "79127", "date": "05 Mar 2021", "name": "Diego E. Gutiérrez Gregoric", "expertise": [ "Reviewer Expertise Malacology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe present study focuses on an invasive alien species with potential effects on human health and agriculture. The population and parasitological analysis in urban areas of Cartagena, Colombia is highlighted, in such a way as to evaluate the role of these snails as potential transmitters of parasites. As the authors postulate, these studies must be constant, since in Colombia itself parasites have already been registered in this snail. With regard to data collection, the estimate of density is not clear, nor is the number of samplings. Did they go to the same site more than once? Is a site the same as a house? These points should be better clarified in the methodology and in the results tables.\nMinor Comments\nKeywords: Achatina fulica must go in italic.\n\nIntroduction: The authors mention the first citation of Achatina fulica in Colombia for 2010, however in 2009 it had already been cited by Borrero et al. (2009, Tentacle N ° 17).\n\nIn Table 1 you must indicate if it is the total number of individuals collected.\n\nFor further parasitological studies on snails, the authors should use other techniques for the observation of larvae, in addition to those used here (see Valente et al 2017, Veterinary Parasitology).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6459", "date": "18 Mar 2021", "name": "Eder Cano Pérez", "role": "Author Response", "response": "Dear reviewer, Thank you for the comments and suggestions to our manuscript." } ] } ]	1	https://f1000research.com/articles/10-77
https://f1000research.com/articles/10-226/v1	22 Mar 21	{ "type": "Software Tool Article", "title": "CytoBrowser: a browser-based collaborative annotation platform for whole slide images", "authors": [ "Christopher Rydell", "Joakim Lindblad", "Christopher Rydell" ], "abstract": "We present CytoBrowser, an open-source (GPLv3) JavaScript and Node.js driven environment for fast and accessible collaborative online visualization, assessment, and annotation of very large microscopy images, including, but not limited to, z-stacks (focus stacks) of cytology or histology whole slide images. CytoBrowser provides a web-based viewer for high-resolution zoomable images and facilitates easy remote collaboration, with options for joint-view visualization and simultaneous collaborative annotation of very large datasets. It delivers a unique combination of functionalities not found in other software solutions, making it a preferred tool for large scale annotation of whole slide image data. The web browser interface is directly accessible on any modern computer or even on a mobile phone, without need for additional software. By sharing a \"session\", several remote users can interactively explore and jointly annotate whole slide image data, thereby reaching improved data understanding and annotation quality, effortless project scaling and distribution of resources to/from remote locations, efficient creation of \"ground truth\" annotations for methods' evaluation and training of machine learning-based approaches, a user-friendly learning environment for medical students, to just name a few. Rectangle and polygon region annotations complement point-based annotations, each with a selectable annotation-class as well as free-form text fields. The default setting of CytoBrowser presents an interface for the Bethesda cancer grading system, while other annotation schemes can easily be incorporated. Automatic server side storage of annotations is complemented by JSON-based import/export options facilitating easy interoperability with other tools. CytoBrowser is available here: https://mida-group.github.io/CytoBrowser/.", "keywords": [ "virtual microscopy", "digital cytology", "whole slide image", "annotation", "assessment", "visualization", "bioimage informatics", "JavaScript" ], "content": "Introduction\n\nThe acquisition and use of whole-slide images (WSIs) is rapidly growing in research and medical practice1,2. The use of WSIs is often referred to as virtual microscopy, due to the appealing ability to digitally recreate the microscopy experience. WSIs are produced by high-resolution scanning of entire microscopy glass slides. The resulting images are often larger than 100,000 × 100,000 pixels and many gigabytes in size; one single image of a cytological or histological specimen may contain more than 100,000 cells. The ability to inspect and annotate WSIs and therefrom derive results has become a crucial component of many bioimage analysis workflows and is also making headway into modern clinical practice. The sheer size of WSI data, where a single image may be larger than the available RAM memory of a standard workstation, is problematic for many standard visualization tools, imposing a need for specialized software solutions.\n\nThere is lately a strong desire within the scientific community towards increased use of open and collaborative solutions and the development of open-source software (OSS)3. A strong argument for the open philosophy is that it efficiently reduces the risk for vendor lock-in situations, where a customer using a proprietary product or service cannot easily transition to another technology due to incompatibilities, inefficient processes, or contract constraints.\n\nA variety of OSS project that support WSIs exist, examples include caMicroscope4, Digital Slide Archive5, Sedeen viewer6, TissUUmaps7, Cytomine8, OMERO9, and QuPath10. The vast majority of existing WSI tools are developed with histology in mind, whereas cytology is only recently gaining increased attention.\n\nOne specificity of cytopathological analysis, as compared to tissue based histopathological analysis, is the need for several focus levels. Where a tissue sample provides a reasonably flat surface, cells of a cytological sample are distributed in a layer of a certain thickness, from a few micrometers for a liquid-based sample, up to several tens of micrometers for a smear. The depth of field of a high resolution light microscope is often below 1 µm, and microscopic analysis of cytological specimen typically involves adjusting focus individually for each cell. A skilled cytopathologist utilizes the limited depth of focus as a way to separate overlapping cells and also to get a 3D feeling of the sample. To accommodate this property in virtual microscopy, it is common to acquire several focal layers of a specimen and create a so called z-stack. Several studies have examined the importance of z-stacking for whole slide imaging; Hanna et al.,11 conclude that z-stacks offer a superior mechanism for overcoming focusing problems encountered with digital cytology slides, while Lu et al.,12 demonstrated that even such a small deviation from the in-focus plane as 0.8 µm may have a clear negative impact on deep learning-based classification performance. Visual assessment of scanned samples requires easy interactive inspection at multiple resolutions and focus levels, but only very few of the above mentioned OSS solutions offer smooth and fast handling of z-stacks, where changing focus is as easy as in a light microscope.\n\nA great benefit of virtual microscopy over optical microscopy is the possibilities for sharing and collaboration. Digitized WSIs can be made accessible via the Internet, thereby enabling efficient usage of distributed resources and reaching remote parts of the world. Tools for interactive visualization and collaborative assessment of WSIs facilitate improved understanding as well as increased quality of analysis, where several experts may share experiences. Collaborative tools for WSI annotations are also gaining popularity in medical education; in a mixed methods trial13 it was found that collaborative WSI annotation provided superior understanding of key microscopic features by senior medical students as compared to individual WSI annotation.\n\nDespite the growing number of tools for WSI visualization and annotation, we find that there is a lack of light-weight, easy to use OSS tools which are well suited for collaborative annotation of cytological data. The majority of existing tools are directed towards histopathology, with different needs and requirements. Aiming to gather the experience and knowledge of experts from all over the globe, an easy to use, browser-based solution, which may be directly utilized on any computer or tablet without need for software installation, is highly desired. The ability for several users to simultaneously view and comment the same sample improves understanding and analysis quality. The CytoBrowser software, presented here, aims to meet all of these needs and desires. It delivers easy and flexible access to WSIs through a browser-based interface, see Figure 1 for an example view. CytoBrowser aims to well replicate the optical microscope feeling, with good support for z-stacks. A growing set of annotation tools are well suited for cytological data. Efficient collaboration is achieved through shared views, with option to follow the view of another user. Annotations may be added and commented on concurrently by several users. The software, completely in JavaScript and Node.js, is light-weight and very easy to install and run. CytoBrowser is typically hosted as a web service, but may also be used locally. We share the source code of CytoBrowser openly and freely under the GNU General Public License v3.0, with the hope that a growing community of users will jointly contribute to its further improvement.\n\nThe user has just placed a marker on a cell nucleus, indicating the Bethesda grade HSIL (High-grade squamous intraepithelial lesion). An overview of the WSI is presented in the lower left part of the screen, with the current zoomed-in viewport indicated as a small red rectangle.\n\n\nMethods\n\nCytoBrowser is a fork and extension of TissUUmaps7 (GPLv3 license), which in turn builds on the OpenSeadragon14 (BSD-3-Clause license) web-based viewer for high-resolution zoomable images. Although TissUUmaps is a very capable tool for visualization of cytology WSIs, it is developed with tissue analysis in primary focus and the user interface and tools are not always ideal for cytological data. CytoBrowser instead puts cytological analysis in the main focus. An important feature for cytological data is efficient handling of z-stacks (currently not supported in TissUUmaps); CytoBrowser delivers fast browsing through focus levels by use of transparent layers. CytoBrowser also allows z-level differentiated annotations, useful, e.g., for annotating overlapping cells.\n\nThe development of CytoBrowser is aimed towards ease of use by cytotechnologists and cytopathologists on a global scale. Implemented tools for shared multi-user sessions facilitate efficient remote collaboration, including options for joint-view visualization and collaborative annotation and assessment of very large datasets. CytoBrowser delivers this through a web-browser interface which is functional on any modern computer or tablet, facilitating efficient utilization of distributed resources. A demonstration setup of CytoBrowser running on several devices (Microsoft Edge browser under Windows, Firefox browser under Linux, Chrome browser on Android tablet and phone) is shown in Figure 2. The multi-user design of CytoBrowser offers concurrent access to samples, where different experts may assess, comment on, or edit each others annotations, or where an educator may, e.g., show students different medically relevant examples.\n\nThe tablet and the laptop in the foreground follow the view displayed on the larger monitor in the background, while the phone is showing a somewhat larger view. The user of the laptop in the foreground is adding a text comment to the upper left marker, while the (followed) user has just placed a HSIL marker on the nucleus in the lower part of the screen. CytoBrowser is compatible with all modern browsers.\n\nThe frontend of CytoBrowser is implemented in JavaScript. The implementation makes use of the Revealing Module15 design pattern to clearly separate different parts of functionality and to make it both easily maintainable and expandable. OpenSeadragon14 is used to display a viewport in the user interface and to dynamically load different parts of the image from the server as needed. The annotations that the user can place on an image belong to one of several annotation classes. By default these classes follow the Bethesda grading system16. Classes are defined in a separate file and can easily be modified to suit the specific task.\n\nCytoBrowser utilizes the Deep Zoom Image (DZI) format for fast rendering of very large WSIs. Currently, each z-layer is provided as a separate DZI file, where a filename of the form imagename_zNNN.dzi indicates an image with name ’imagename’ at z-level ’NNN’, and where ’NNN’ is any number, typically an offset in nanometer.\n\nThe backend of CytoBrowser is run in Node.js17, with a web server implemented with the Express18 module. The server provides HTTP endpoints for getting information about the available images as well as for writing and reading annotation data in server-side storage. While these endpoints are mainly intended to be used by the CytoBrowser client, they could also be used to easily access this information from other applications. The server also provides a WebSocket endpoint for full-duplex communication between the client and server to facilitate the collaboration functionality. This communication is done using JSON messages. The only mandatory field in a such a message is the type field, which informs the recipient how to process any other fields that may be present.\n\nWhen a user opens an image in CytoBrowser, they either select an existing set of annotations to work on or start a new set. Different sets of annotations for the same image may, e.g., originate from different experts or be imported from a machine learning-based classification. The annotation set also defines a collaboration session, consisting of all users currently working on the same annotation set, much like users collaborate on the same document in web-based applications such as Google Docs19. By encoding the current state of the interface, including the active image and annotation set, but also viewport location and zoom level, in the dynamically updated URL, it becomes very easy to invite colleagues to discuss a sample by simply sharing the active URL. In the same manner, it is easy to return to a previous image location through a stored browser bookmark.\n\nThe protocol implemented for collaborations has been created with robustness in mind. The backend stores a canonical copy of the shared data, including information about the participating users, the active image and the placed annotations, that the clients request when entering a collaboration. The backend also verifies that clients are displaying the correct image. When data is altered by a connected client, only the alteration is broadcast to other collaborators.\n\nFor additional details regarding the implementation, we refer the reader to the implementation.md file of the software.\n\nOpening images stored on the server is easy; available images are presented to the user as a gallery of thumbnails, see Figure 3 for an example. By default these thumbnails display a lower-resolution version of the full image, but by hovering over a specific entry the user is presented a zoomed-in view from the middle part of the image. After selecting an image, the user is prompted to either start a new annotation session or to continue on a previously created.\n\nCytoBrowser enables placing several different kinds of annotations on an image. Markers can be placed by clicking a single point, rectangular regions can be placed by clicking two opposite corners, and polygonal regions can be placed by clicking each vertex, see Figure 4 for an example of the latter. While these three annotation tools are currently present in CytoBrowser, the system is designed to allow easy addition of new tools.\n\nOnce annotations are placed, there are different ways of interacting with them. They can be moved or deleted using the mouse. By right-clicking an annotation, the user gets access to more actions in a context menu. Here they can change the class assigned to the annotation or leave text comments on it to provide information that is not captured by the class label alone. In Figure 2, the user of the laptop in the foreground is adding such a comment.\n\nThree different options for saving and loading annotation data are provided. The easiest option is for the user to rely on the autosave functionality of CytoBrowser, where the current annotation set is continuously stored on the hosting server. The second option is to manually store the data on the server. For this option, we also provide a simple versioning system. If a file is saved to a directory that contains a file with the same name, the user gets the option to either rewrite the latest version of the file or to create a new version of it. The user is then able to choose which version of a file to load when browsing the server files. The third option is to store the annotation set on the local machine of the user, using the “import” and “export” buttons in the interface. For all three options, the annotations are stored as JSON files, making the data easily transferable between different tools. This enables the annotations to be used for data selection or training of machine learning methods. Inversely, CytoBrowser may be used to visualize the classification or segmentation results generated by other tools.\n\nWe believe that easy collaboration between experts from different fields is important for an efficient workflow. For CytoBrowser, we have implemented functionality that enables such collaboration to be performed remotely, where two or more users can browse and annotate the same image together in a shared session. Any changes made by one user will be seen by the other users in real-time. Users can see each other’s mouse cursors in the viewport, and they have the option to lock their view to follow that of another user. This may be useful, e.g., in cases where an expert from one domain needs to visually explain something about the data to an expert in another domain. This capability may thus enhance the transfer of knowledge between different fields and simplify multidisciplinary research.\n\n\nUse cases\n\nTo download and install the software, follow the instructions at https://mida-group.github.io/CytoBrowser/. It is very easy to set up a local CytoBrowser server to be accessed from the same machine (localhost), or, e.g., through an SSH tunnel. For larger and more persistent use cases, we recommend to rely on one of popular open-source web servers, such as Apache and Nginx, as a secure loadbalancing frontend for the server.\n\nExample Bash scripts, demonstrating how to download and convert publicly available images in Zeiss (.zvi) and Hamamatsu (.ndpi) format into the Deep Zoom Image (.dzi) format, suitable for CytoBrowser, are provided together with the software in the examples directory. Software packages which may be useful for converting WSIs include libvips, Bio-Formats, and NDPITools.\n\n\nConclusions\n\nWe present CytoBrowser, an OSS developed in response to the request for light-weight, open, flexible, and easy to use tools for collaborative visualization, annotation and assessment of whole slide image data. CytoBrowser is developed particularly with cytological analysis on mind, where efficient handling of z-stacks is a central requirement. The combi- nation of a JavaScript based browser component and a Node.js driven server-side component enables fast access to large image data together with easy and flexible storage and sharing of annotations. Being a tool particularly designed for collaborative interactive multi-user access, it facilitates knowledge exchange and alleviates interdisciplinary research.\n\nIn addition to being a much desired resource for the creation of large annotated datasets, crucial for training and evaluation of machine and deep learning-based approaches to bioimage analysis, as well as the visualization and qualitative evaluation of the results of such methods, we also envision that CytoBrowser may be a useful tool for distributed education, quality improvement, competency assessment, proficiency testing, and performance evaluation20.\n\nBy delivering a powerful virtual microscope environment which is easily accessed through a web-browser interface and functional on any modern computer or tablet, we hope that CytoBrowser, facilitating efficient utilization of distributed resources and expert knowledge, may contribute towards fair and universally accessible health-care.\n\n\nSoftware availability\n\nSoftware available from: https://mida-group.github.io/CytoBrowser/\n\nSource code available from: https://github.com/MIDA-group/CytoBrowser\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.458276021\n\nLicense: GNU General Public License v3.0", "appendix": "Acknowledgements\n\nWe thank Leslie Solorzano and Prof. Carolina Wählby for their support in forking TissUUmaps.\n\nThis publication was supported by COST Action NEUBIAS (CA15124), funded by COST (European Cooperation in Science and Technology).\n\n\nReferences\n\nAeffner F, Zarella MD, Buchbinder N, et al.: Introduction to digital image analysis in whole-slide imaging: a white paper from the digital pathology association. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMartel AL, Hosseinzadeh D, Senaras C, et al.: An image analysis resource for cancer research: Piip—pathology image informatics platform for visualization, analysis, and management. Cancer Res. 2017; 77(21): e83–e86. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSolorzano L, Partel G, Wählby C: TissUUmaps: Interactive visualization of large-scale spatial gene expression and tissue morphology data. Bioinformatics. 2020; 36(15): 4363–4365. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMarée R, Rollus L, Stévens B, et al.: Collaborative analysis of multi-gigapixel imaging data using cytomine. Bioinformatics. 2016; 32(9): 1395–1401. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAllan C, Burel JM, Moore J, et al.: OMERO: flexible, model-driven data management for experimental biology. Nat Methods. 2012; 9(3): 245–253. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBankhead P, Loughrey MB, Fernández JA, et al.: QuPath: open source software for digital pathology image analysis. Sci Rep. 2017; 7(1): 16878. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHanna MG, Monaco SE, Cuda J, et al.: Comparison of glass slides and various digital-slide modalities for cytopathology screening and interpretation. Cancer Cytopathol. 2017; 125(9): 701–709. PubMed Abstract \| Publisher Full Text\n\nLu J, Sladoje N, Stark CR, et al.: A deep learning based pipeline for efficient oral cancer screening on whole slide images. In International Conference on Image Analysis and Recognition. 2020; 249–261. Springer. Publisher Full Text\n\nSahota M, Leung B, Dowdell S, et al.: Learning pathology using collaborative vs. individual annotation of whole slide images: a mixed methods trial. BMC Med Educ. 2016; 16(1): 311. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOpenSeadragon: An open-source, web-based viewer for high-resolution zoomable images, implemented in pure JavaScript, for desktop and mobile. Accessed on: Feb. 2021. Reference Source\n\nThe revealing module pattern in javascript. Accessed on: Feb. 2021. Reference Source\n\nNayar R, Wilbur DC: The Bethesda system for reporting cervical cytology: definitions, criteria, and explanatory notes. Springer, 2015. Reference Source\n\nNode.js: A javascript runtime built on Chrome’s V8 JavaScript engine. Accessed on: Feb. 2021. Reference Source\n\nExpress: Fast, unopinionated, minimalist web framework for Node.js. Accessed on: Feb. 2021. Reference Source\n\nAnnouncing Google Workspace, everything you need to get it done, in one location. Google Cloud Blog. Accessed on: Feb. 2021. Reference Source\n\nQuinn AM, Minhajuddin AT, Hynan LS, et al.: Agreement between cytotechnologists and cytopathologists as a new measure of cytopathologist performance in gynecologic cytology. Cancer Cytopathol. 2017; 125(7): 576–580. PubMed Abstract \| Publisher Full Text\n\nRydell C, Lindblad J: MIDA-group/CytoBrowser: Bugfix release v1.0.1, URL location encoding (Version v1.0.1). Zenodo. http://www.doi.org/10.5281/zenodo.4582760" }	[ { "id": "81858", "date": "08 Apr 2021", "name": "Simon Nørrelykke", "expertise": [ "Reviewer Expertise Image analysis. Biophysics." ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors introduce CytoBrowser, an open-source, server-client, browser-interface solution for collaboratively viewing and annotation of large microscopy image data. CytoBrowser is a fork of TissUUmaps, adding handling of z-stacks incl. z-level dependent annotations.\nThe intended audience are cytotechnologist and cytopathologists, as opposed to histopathologists. CytoBrowser also aims to be used in a (remote) teaching setting, through the use of synchronisation between the teacher's/expert's browser-view and the students.\nOverall the paper is well written with few if any typographical or grammatical errors and lays out its goal and ambitions in a rather clear fashion.\nHowever, the overall feeling is that CytoBrowser is not ready to be shared with the world as it is missing proper installation instructions, a user-manual, and a demo-site. It feels like a local lab-tool that the authors are taking steps to make more broadly accessible, which is commendable, but there is still some way to go.\nOBSERVATIONS ON WRITING STYLE:\nThe Introduction is comparatively long, reads somewhat like a sales pamphlet, and includes most of the information repeated in the very brief Methods.\nThere is a tendency of mixing up turns of phrase, e.g. \"making headway into\", \"There is lately\", \"in the main focus\", and \"on mind\"; which are probably better put as \"making its way into\" or \"making headway in\" etc.\nVariations of \"easy\" (easy, easily, easiest) appear 24 times - in most instances the word could be omitted for a more sober delivery, losing no information.\nRECOMMENDATIONS:\nMake a web-server demo-version available so the potential user can try it out without having to install the full solution.\nCompare to commercial solutions - open source is not the only player out there. How does is stack up against e.g. Arivis (https://imaging.arivis.com/)?\nImprove the installation instructions by having someone try to follow them: I gave it 30 minutes and failed to get it to run (details below).\nMake it run on MacOS as well: Many, if not most, biologists use Macs, not Windows, and definitely not Linux.\nBe explicit about which other softwares CytoBrowser can directly import/export annotations from/to. Does it play well with e.g. ilastik or Imagej (see here for some ref: https://f1000research.com/articles/9-1248 incl. Jan Eglinger's referee report)\nBe explicit about what image formats are natively supported - it seems only .dzi and the user have to make the conversion using some of your provided scripts or find another way. This is fine, but be very upfront about this\nWrite a proper user-manual either in GitHub or in https://readthedocs.org/ - this is the accepted standard for useful open-source software looking for users. It is work and it is boring, but it is neccessary if you want to have users outside your own lab.\nLITTLE THINGS:\nThe figures online and in PowerPoint are not vector graphics and do not allow zooming to a useful level. PDF file is fine.\nINSTALLATION STEPS TAKEN:\nInstallation on Ubuntu 20.04.2 LTS running on a Thinkpad. Notice that the Linux commands are different from and additional to the ones you give in your instructions, in order to get just some of the way. I am not saying it cannot be done, I am saying that it would likely take more time than is usual for shared open-source code and defintely more than a test-user can be expected to invest.\n#+begin_src git clone https://github.com/MIDA-group/CytoBrowser.git cd CytoBrowser/\nsudo apt install npm\nsudo apt install curl sudo apt-get install -y nodejs curl -fsSL https://deb.nodesource.com/setup_14.x \| sudo -E bash -\nnpm install express --save npm install\nmkdir data mkdir storage mkdir collap_storage\n#+end_src\nDownloaded the Zeiss example images and moved them to ./data\nRunning (failing to run): #+begin_src simon@simon-ThinkPad-P1-Gen-2:~/Desktop/CB/CytoBrowser$ node cytobrowser.js localhost 8080 CytoBrowser server listening at http://127.0.0.1:8080 /home/simon/Desktop/CB/CytoBrowser/server/availableImages.js:142\n\nlet names = dir.map(name => name.match(nameEx)).flat();\n\n^\nTypeError: dir.map(...).flat is not a function\n\nat fs.readdir (/home/simon/Desktop/CB/CytoBrowser/server/availableImages.js:142:57)\n\nat FSReqWrap.args [as oncomplete] (fs.js:140:20) #+end_src\nCONCLUSION: Currently I cannot approve for indexing as I could not verify that the software actually works or does any of the wonderful things described in the manuscript: The test of reproducibility failed! If the relevant recommendations re. documentation, installation instructions, and a test-server are followed, I would be happy to recommend indexing (after verifying that the software works) - any stylistic and linguistic idiosyncrasies are entirely the choice of the authors and will not influence my decision.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Partly\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? No\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "6560", "date": "13 Apr 2021", "name": "Joakim Lindblad", "role": "Author Response", "response": "Response to Reviewer Report #1 (S. Nørrelykke) Reviewer: “Overall the paper is well written with few if any typographical or grammatical errors and lays out its goal and ambitions in a rather clear fashion.” Response: We are glad to read that the reviewer appreciates our aim towards clarity of presentation. Reviewer: “However, the overall feeling is that CytoBrowser is not ready to be shared with the world as it is missing proper installation instructions, a user-manual, and a demo-site. It feels like a local lab-tool that the authors are taking steps to make more broadly accessible, which is commendable, but there is still some way to go.” Response: We agree with the reviewer that a user-manual and a demo-site are certainly desirable additions that will further improve accessibility to the tool. However, we strongly disagree with the reviewer that an absence of such renders it inappropriate to be shared. Yes, it is a “local lab-tool”, which we have developed based on our own needs, and yes, we feel that it may be useful for a wider community and therefore we wish to inform a broader audience about its existence and invite them to try, use, and possibly contribute to its further development in the spirit of FOSS. The alternative, to not publish the tool before we have found resources to host a demo-site and to write a separate user-manual (to complement the existing help pages of the software), seems as a rather unneeded delay. Further, we consider the installation instructions, which assume an ability to install required auxiliary software, to be sufficiently detailed for the envisioned users of the server side of the tool (see also later part of this response). We emphasize that once set up, users of the browser based interface (including cytotechnologist and cytopathologists) need not perform any installation at all. Regarding Observations on writing style: We appreciate the reviewers opinions and will take them into consideration when updating the manuscript; we will await other reviewer’s responses to spare them from reviewing a moving target. Regarding Recommendations: We hope to find resources to host a demo web-server. We do not consider that to be a requirement for publication though. On a comparison with commercial solutions, we do not consider a closed proprietary solution a reasonable alternative, as clearly explained in the paper. “Make it run on MacOS” – All parts of the software runs well on MacOS. (We wonder how the reviewer reached the (incorrect) conclusion that it does not.) “Many, if not most, biologists use Macs” – Additionally, the end user may run the interface on any browser, in any OS (we believe this is made clear in the manuscript). “Be explicit about what image formats are natively supported” – This is clearly stated at several places in the text, including very explicitly in the “Use cases”. “The figures online and in PowerPoint are not vector graphics” – This is due to F1000Research, we will ask them to improve this; we recommend the PDF version where figures are of higher resolution. INSTALLATION STEPS TAKEN: Prompted by the reviewer’s attempt, we performed an installation on a clean Ubuntu 20.04.2 LTS. This confirmed that the steps listed in the installation instructions are accurate and work as intended. The instructions explicitly point to external sources on how to install an up-to-date Node.js (version 12 or later required). For the reviewer’s specific case of Ubuntu (which ships with v10 of Node.js, despite v12 being out since April 2019) we recommend to verify that the Node.js update actually succeeded, by typing ‘node -v’ in a terminal and confirming the correct version. The error performed by the reviewer is that the following two operations were executed in the wrong order (see also the output of the ‘setup_14.x’-step): curl -fsSL https://deb.nodesource.com/setup_14.x \| sudo -E bash - sudo apt-get install -y nodejs The below short list of steps perform the complete installation on a clean Ubuntu 20.04.2 LTS (these steps also match the installation instructions): #+begin_src #curl needed for the Node.js update, libvips needed for conversion of data to .dzi sudo apt -y update && sudo apt -y install git npm curl libvips-tools #install a newer node.js for Ubuntu (v12 works as well) curl -fsSL https://deb.nodesource.com/setup_14.x \| sudo -E bash - sudo apt-get install -y nodejs #the CytoBrowser installation starts here git clone https://github.com/MIDA-group/CytoBrowser.git cd CytoBrowser npm install #start the CytoBrowser server node cytobrowser.js localhost 8080 #In a separate terminal we download and convert some data using one of the provided example scripts: cd CytoBrowser ./examples/Hamamatsu-OS-2.sh #downloads, converts, and opens a browser window #+end_src If there are any problems, we recommend the Reviewer to post an ‘Issue’ on GitHub, which is the standard and recommended way to handle such events." }, { "c_id": "6577", "date": "14 Apr 2021", "name": "Joakim Lindblad", "role": "Author Response", "response": "LITTLE THINGS: Reviewer: \"The figures online and in PowerPoint are not vector graphics and do not allow zooming to a useful level.\" Thanks for pointing this out. We have contacted F1000Research about the issue and they have now (this morning) replaced the web images (and pptx) with higher resolution files." } ] }, { "id": "81855", "date": "30 Apr 2021", "name": "Peter Bankhead", "expertise": [ "Reviewer Expertise image analysis", "digital pathology", "open-source software" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper introduces CytoBrowser, an open-source solution for viewing and annotating whole slide images and other microscopy data. CytoBrowser is a fork of TissUUmaps, adding new functionality to support z-stacks and collaborative annotation.\nCytoBrowser looks like it could be very useful for a limited number of specific applications already, and potentially many more with additional development. I believe the paper would be strengthened considerably by describing in more detail the applications where CytoBrowser has been used.\nFurther documentation and information about future development plans would also be essential if the software is to be widely adopted. To make use of Cytobrowser's collaborative features (which seem a major strength), it would be necessary to set up a server - requiring some level of computing knowledge, effort and resources. For this reason, I think it is important to make the benefits of using the software as clear as possible, and the process for using it as straightforward as possible; without this, I am not sure why a potential user would make the effort. A demo server, video demos, more illustrated documentation, and more detail within this article would all help with this.\nSpecific points and questions\n'A variety of OSS project that support WSIs exist, examples include caMicroscope4, Digital Slide Archive5, Sedeen viewer6, TissUUmaps7, Cytomine8, OMERO9, and QuPath10. The vast majority of existing WSI tools are developed with histology in mind, whereas cytology is only recently gaining increased attention.' - I think SlideRunner (https://github.com/DeepPathology/SlideRunner) should be included. Since there is no real direct comparison, it is not clear what the benefits of CytoBrowser are over the others for any applications. A table comparing relevant features could address this.\n'The majority of existing tools are directed towards histopathology, with different needs and requirements.' - Other than z-stack support (which at least some of the listed software provides), I'm not sure what these different needs and requirements are - at least in terms of the annotation tools (points, rectangles, polygons) provided by CytoBrowser.\n'We present CytoBrowser, an OSS developed in response to the request for light-weight, open, flexible, and easy to use tools for collaborative visualization, annotation and assessment of whole slide image data.' - I suggest swapping 'request' for 'need', or else elaborating upon who requested it and for what purpose.\n'CytoBrowser provides a web-based viewer for high-resolution zoomable images and facilitates easy remote collaboration, with options for joint-view visualization [...]' - It's not obvious to me what 'joint-view visualization' means (I assume multiple users seeing the same region?).\n'The multi-user design of CytoBrowser offers concurrent access to samples, where different experts may assess, comment on, or edit each others annotations, or where an educator may, e.g., show students different medically relevant examples.' - Has CytoBrowser been used in education, or would it require further development? Can access and annotation be controlled (i.e. to restrict changes students can make)? In a sense, any platform that allows image viewing might be used in education. If there are specific strengths in using CytoBrowser for this, these should be specified.\n'The protocol implemented for collaborations has been created with robustness in mind. The backend stores a canonical copy of the shared data, including information about the participating users, the active image and the placed annotations, that the clients request when entering a collaboration. The backend also verifies that clients are displaying the correct image. When data is altered by a connected client, only the alteration is broadcast to other collaborators.' - I believe the robustness claim needs supporting evidence, particularly given the focus on being lightweight and using json files for storage. Has CytoBrowser been stress-tested, with a large number of concurrent users? Is there an expected limit to the number of concurrent users?\n'Inversely, CytoBrowser may be used to visualize the classification or segmentation results generated by other tools.' - How? Are any open standards supported for importing annotations, or must they be converted to a CytoBrowser-specific format? How scalable is this, e.g. does it support thousands or even millions of 'objects'/regions? Are rasterized segmentation results supported (i.e. overlapping another image, rather than annotations)?\n'CytoBrowser delivers fast browsing through focus levels by use of transparent layers.' - What are 'transparent layers' here?\n'While these three annotation tools are currently present in CytoBrowser, the system is designed to allow easy addition of new tools.' - How? Is this documented?\n'CytoBrowser is a fork and extension of TissUUmaps7 (GPLv3 license)' - Is there a justification for forking TissUUmaps, rather than contributing to the original project? How will both be maintained in the future? Is there any intention to incorporate future improvements to one software within the other?\n'We share the source code of CytoBrowser openly and freely under the GNU General Public License v3.0, with the hope that a growing community of users will jointly contribute to its further improvement.' - Given that fulfilling the potential of CytoBrowser (as outlined in the paper) would seem to require further development, I would like to understand if the authors intend to further develop the software themselves. Community development would require central maintenance; without clear evidence of active ongoing support it is difficult to see how this hope can be fulfilled.\nUse of CytoBrowser\nI was able to successfully run CytoBrowser locally on a Mac. There were a two minor complications, and one possibly bigger limitation:\nTo run the example scripts, I needed to install wget and libvips (via homebrew), as well as finding, downloading & unzipping bftools. Further documentation, with links, would help others do this.\n\nNo images are available when using Safari locally, but without explanation - it simply appears to be broken. With Chrome and Firefox it works, but some form of notification when Safari fails would be helpful. This is not entirely compatible with the statement 'CytoBrowser is compatible with all modern browsers.'\n\nThe proposed ZVI example file is extremely dark. Since .dzi involves conversion to JPEG, colours and contrast seem to be 'fixed' at the conversion step - without any option to adjust these through the UI. This seems like a significant limitation for non-RGB images. Is there a solution?\n\nThe need to generate .dzi datasets is a barrier to testing the software (which I imagine is harder on Windows). Providing a relevant dataset (ideally for cytology) without a need for conversion would help overcome this.\nSome further comments on using the software:\nI don't recognize the format of the JSON export; is it custom for CytoBrowser? For interoperability, I think a standard such as GeoJSON would be strongly preferable.\n\nThe 'Usage instructions' within the UI are very helpful, although in some cases it would be helpful to have instructions closer to the relevant command, e.g. via tooltips.\n\nShortcuts are unexpected (e.g. c, v, b to change annotation tool). Shift + mouse wheel is nice to rotate, but I expected the rotation direction to change with the scroll direction.\n\nI find the Session and Collaboration interfaces confusing. I found myself inadvertently creating many new sessions when exploring whether I can make changes to the current one (e.g. to set its name). I think 'Session name' should be above 'Username' under the 'Collaboration' window, but it's not clear to me what is the impact of changing either, nor whether 'Change session' means 'change the properties of the session' or 'switch to a different session'.\n\nThe use of separate .dzi datasets for z-slices seems a bit awkward; it would help if these could at least be arranged within the same subdirectory (rather than all inside one 'data' directory) for better data organization.\n\nClicking on 'Session' at the top of the screen allows me to change the user, but clicking on 'User' does not.\n\nThe collaborative features of CytoBrowser sound like a particular strength of the platform, however I was unable to test these.\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? No", "responses": [] } ]	1	https://f1000research.com/articles/10-226
https://f1000research.com/articles/10-225/v1	22 Mar 21	{ "type": "Study Protocol", "title": "Bibliometric analysis on workplace cyberbullying: study protocol", "authors": [ "Yun Jin Kim", "Linchao Qian", "Muhammad Shahzad Aslam", "Yun Jin Kim", "Linchao Qian" ], "abstract": "Cyberbullying behaviour is an international public health concern all around the world due to the increasing trend of working from home during COVID-19. The prevalence of workplace cyberbullying behaviour (WCB) has been shown to be increased prior to COVID-19 among allied health professionals, such as nurses and trainee doctors. There has been a lack of bibliometric analysis on scientific publications concerning this subject; therefore, the current articles presents a protocol for bibliometric analysis of WCB. An indicator-based search will have carried out from documents on PubMed and Scopus to retrieve data from primary peer-reviewed WCB research articles using relevant keywords. Articles that involve WCB research will be included in the analysis. The dataset will identify documents all around the world, and data will be validated using the VAKS assessment tool. Analysis will be carried out by comparing the relationship among institutions, authors, countries and keywords. The dataset will be publicly accessible in the Zenodo repository. There will be no involvement of human participants; therefore, the current research does not require an ethical review. Results will be publish in a peer-reviewed journal and at related conferences", "keywords": [ "Workplace", "Cyberbullying", "health informatics", "Information management", "Human resource management", "Organisational development" ], "content": "Introduction\n\nCyberbullying is defined as “an aggressive, intentional act carried out by a group or individual, using electronic forms of contact, repeatedly and over time against a victim who cannot easily defend him or herself”.1 The past definition has a limited scope, not applicable to online workplace harassment and focussed on adolescents only. However, recent research concludes the definition of workplace cyberbullying behaviour (WCB) as “[involving] perceived unwanted or aggressive behaviour(s) perpetrated at any time through electronic media, that may harm, threaten, or demoralise the intended target(s) of this behaviour(s)”.2\n\nCyberbullying has become a critical youth issue worldwide.3 A recent death of 25-year-old Sulli Choi (October 14, 2019), who was a famous South Korean pop star has raised issues of growing cyberbullying. Before her death, Sulli had been long harassed by cyberbullying, including hate speech, stalking, and threats.4,5 Ministry of Education of the Republic of Korea and the National Research Foundation of Korea has recently funded several research projects on cyberbullying6 and workplace cyberbullying.7\n\nThere is a tremendous increase in the usage of mobile technology in the workplace. Several studies consider the positive influence of employees' in the perceived improvement of work performance,8 yet the extensive usage of mobile phone results in significant mental health issues (i.e. depression, sleep disturbance, stress and anxiety).9 The era of COVID-19 brings several challenges due to increased working from home (WFH), and may have a negative consequence on mental health.10,11\n\nA bibliometric study is an in-depth descriptive study investigating the characteristics of publications, helps in the identification of research gaps, and highlights trends in relevant research domains.12 The inclusion of visualisation and data mining technique demonstrates a clear picture of a specific topic. The current protocol intends to discuss the research trends and characteristics published in WCB research. The study will determine the number of research publications; classify studies into themes and sub-themes; identify the research gap in WCB research; and demonstrate trends in author affiliations and institutional collaborations using qualitative methods.\n\nThis study aims to describe the literature available on Scopus and PubMed concerning workplace cyberbullying.\n\n\n\n1. Explore the bibliometric indicators on workplace cyberbullying.\n\n2. Describe the trends between years (article published) correlated with the number of citations obtained.\n\n3. Describe correlations between authors (in numbers), institutions, country collaborations, funding, and topics addressed.\n\n\n\n1. What significant issues have been discussed among selected articles on workplace cyberbullying?\n\n2. Which articles and authors are highly cited in the topic of workplace cyberbullying?\n\n3. Which universities/institution/organisation and funding agencies are involved in collaborating research on workplace cyberbullying?\n\n\nMethods\n\nThe proposed study will investigate bibliometric indicators, with an emphasis on the quantitative synthesis of scholarly publication data. The research will evaluate the application of output and impact indicators for the evaluation of research published. Bibliometrics is essential for research evaluation, and there has been an increasing trend observed in this type of research since the mid-1980 in the field of natural and life sciences. It is additionally interesting for social sciences’ scientists because many methods used in bibliometrics are from the social sciences’ disciplines.13\n\nThe researcher will use PubMed and Scopus databases to identify related papers. PubMed is known as a wide-ranging database in the field of mental health research14 and includes research on public health issues. PubMed is considered as a free text data source and can be used as a public health surveillance platform.15 Scopus is an abstract and indexing database with full-text links that is produced by the Elsevier Co.16 It is a fast method to identify global scientific collaborations.17 Scopus used CiteScore as a journal based matrix that comprises of 22,256 journals, compared with the Journal Impact Factor (JIF), which only includes 11,365 journals.18 The selection of CiteScore is based upon the inclusion of a broader range of article types or documents.19\n\nThe focal theme of the current study will investigate the bibliometric trends in workplace cyberbullying in healthcare professionals. The current research will examine articles published since the beginning of research by scholars till December 2021. 2\n\nThe study will cover academic articles published in PubMed indexed and Scopus indexed journals only. The current scope of articles will be limited to communication health, social health and mental health perspectives.\n\nThe researchers will perform a wide-ranging search for related publications that consists of all field terms and phrases relevant to cyberbullying and the workplace. The access to the article, i.e. Open Access format with full-text availability, will be are consider to assess the visibility of articles.\n\n\n\n1. “Cyberbullying” AND “workplace” AND “office AND “work” AND “workroom” AND “workshop” AND “workstation” AND “place of work” AND “workspace” AND “studio” AND “working area” AND “atelier” AND “shop” AND “headquarters” AND “plant” AND “bureau” AND “department” AND “building” AND “station” AND “branch” AND “showroom” AND “occupation” AND “workforce” AND “workfellow” AND “workmates”\n\n2. “Bullying online” AND “workplace” AND “office AND “work” AND “workroom” AND “workshop” AND “workstation” AND “place of work” AND “workspace” AND “studio” AND “working area” AND “atelier” AND “shop” AND “headquarters” AND “plant” AND “bureau” AND “department” AND “building” AND “station” AND “branch” AND “showroom” AND “occupation” AND “workforce” AND “workfellow” AND “workmates”\n\n3. “Cyberharassment” AND “workplace” AND “office AND “work” AND “workroom” AND “workshop” AND “workstation” AND “place of work” AND “workspace” AND “studio” AND “working area” AND “atelier” AND “shop” AND “headquarters” AND “plant” AND “bureau” AND “department” AND “building” AND “station” AND “branch” AND “showroom” AND “occupation” AND “workforce” AND “workfellow” AND “workmates”\n\n4. “Harassment online” AND “workplace” AND “office AND “work” AND “workroom” AND “workshop” AND “workstation” AND “place of work” AND “workspace” AND “studio” AND “working area” AND “atelier” AND “shop” AND “headquarters” AND “plant” AND “bureau” AND “department” AND “building” AND “station” AND “branch” AND “showroom” AND “occupation” AND “workforce” AND “workfellow” AND “workmates”\n\n5. “Cyberstalking” AND “workplace” AND “office AND “work” AND “workroom” AND “workshop” AND “workstation” AND “place of work” AND “workspace” AND “studio” AND “working area” AND “atelier” AND “shop” AND “headquarters” AND “plant” AND “bureau” AND “department” AND “building” AND “station” AND “branch” AND “showroom” AND “occupation” AND “workforce” AND “workfellow” AND “workmates”\n\nThe researcher will include conceptual studies, empirical analyses, theoretical contributions, methodological studies, data notes, study protocols, scoping reviews, meta-analyses, systematic reviews, narrative reviews, intervention studies, editorials, case studies, commentaries, brief reports and communications retrieved from PubMed and Scopus in the English language from the beginning of the database until December, 2021.\n\nPublications with irrelevant or out of scope topics will be excluded. Magazines, newspapers, articles, books, book chapters, monographs, and conference papers will be excluded from the analysis. The researchers will exclude publications categorised under corrections or retracted.\n\nFor workplace cyberbullying among healthcare professionals, the researchers will extract the following data:\n\n1. Documents type, classified as conceptual studies, empirical analyses, theoretical contributions, methodological studies, data notes, study protocols, scoping reviews, meta-analyses, systematic reviews, narrative reviews, intervention studies, editorials, case studies, commentaries, brief reports and communications.\n\n2. Article access type, classified as Open Access with full-text availability to evaluate the accessibility of relevant documents.\n\n3. Title, name of the first author, name of the corresponding author, h-index of the author (first/corresponding) given by Scopus, number of citations given by Scopus, name of journal/conference, journal ranking according to Scopus (CiteScore), year of publication, name of universities/organisation collaborated, keywords used in related publications and funding availability will be considered as crucial indicators for further scientific evaluation.\n\n4. The top 50 cited papers will be listed.\n\n5. Geographic distribution of publications.\n\n6. Mapping of keywords.\n\nThe following data items will be extracted:\n\n1. Title (Scopus)\n\n2. Name, location and affiliation of first and corresponding author (Scopus)\n\n3. H-index of first and corresponding author (Scopus)\n\n4. Number of Scopus citation (Scopus)\n\n5. Name of journal (Scopus)\n\n6. Journal rank according to Scopus (CiteScore rank)\n\n7. Date of publication, submission and acceptance given by the journal\n\n8. Acceptance time calculated from the journal’s information\n\n9. Number of Universities/Organisation contributed to that publication (Scopus)\n\n10. Name of Universities/Organisation contributed as an author\n\n11. Journal impact factor (Cite Score) according to Scopus 2020\n\n12. Type of Article given by journal or Scopus\n\n13. Major topic/category, the subject area given by Scopus or journal\n\n14. Keyword by journal\n\n15. Major issue addressed will manually collect from the journal.\n\n16. Number of countries involved in collaboration given by the journal.\n\n17. Number of pages and reference provided by Scopus\n\n18. Number of figures and tables\n\n19. Funding and abstract information given by the journal\n\nThe current study will be internally validated by VAKS assessment tool, which is used as a validation tool for qualitative research to assess trustworthiness of qualitative research articles.20\n\nThe researchers will export the data and analyse them using Microsoft Excel 2016 and IBM SPSS Statistics 27. Percentages of Citation analysis, characteristics of authors of exporting articles, Manuscript characteristics will be performed using IBM SPSS Statistics 27 where necessary. The qualitative assessment of indicators will be analysed using Orange software 3.27.1, QDA miner lite and VOSviewer software.\n\nThe current research will not contain documents from preprint servers to avoid the risk of misleading articles that have not been peer reviewed. Articles will be excluded from the list in-case of any conflict observed (i.e. data items) between journal and Scopus indexed articles.\n\nThe proposed study does not directly involve human participants and has been exempt from ethical review.\n\n\nStudy Status\n\nThe current study has not been started yet.\n\n\nDissemination\n\nThe proposed study will identify the bibliometric research trends in workplace cyberbullying behaviour all around the world. Furthermore, the research draws attention to strengths, weaknesses and opportunities in research areas, for instance the type of research being conduct could provide suggestions to researchers on the allocation of research budgets on new themes or subthemes. The researcher proposed to publish the research article in an open access journal. The data will be stored under a publicly accessible Zenodo repository.23 The results will be published in an open access journal after peer-review and will disseminated to the public through scientific conferences (symposium, workshop, or meeting). The published article will be share on scientific social media, such as ResearchGate.24\n\n\nConclusions\n\nUsing bibliographic indicators, the proposed study will identify trends in workplace cyberbullying behaviour research publications, and will examine the extent and pattern of collaboration between researchers globally. A limitation of this study will be language bias due to the selection of English-only articles.\n\n\nData Availability\n\nNo data are associated with this article.", "appendix": "References\n\nSmith PK, Mahdavi J, Carvalho M, et al.: Cyberbullying: its nature and impact in secondary school pupils. J Child Psychol Psychiatry. 2008; 49: 376–385. PubMed Abstract \| Publisher Full Text\n\nD’Souza N, Forsyth D, Tappin D, et al.: Conceptualizing workplace cyberbullying: Toward a definition for research and practice in nursing. J Nurs Manag. 2018; 26: 842–850. PubMed Abstract \| Publisher Full Text\n\nKowalski RM, Giumetti GW, Schroeder AN, et al.: Bullying in the digital age: A critical review and meta-analysis of cyberbullying research among youth. Psychol Bull. 2014; 140: 1073–1137. PubMed Abstract \| Publisher Full Text\n\nLing C, Stringhini G: Examining the Impact of Social Distance on the Reaction to a Tragedy A Case Study on Sulli’ s Death. Assoc Adv Artif Intell. 2020.\n\nYi HY, Cha S: Cyber bullying, star suicides: The dark side of South Korea’s K-pop world\|The Star.Reference Source [Accessed December 12, 2020]\n\nKim H, Han Y, Song J, et al.: Application of Social Big Data to Identify Trends of School Bullying Forms in South Korea. Int J Environ Res Public Health. 2019; 16: 2596. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPark M, Choi JS: Effects of workplace cyberbullying on nurses’ symptom experience and turnover intention. J Nurs Manag. 2019; 27: 1108–1115. PubMed Abstract \| Publisher Full Text\n\nYueh H-P, Lu M-H, Lin W: Employees’ acceptance of mobile technology in a workplace: An empirical study using SEM and fsQCA. J Bus Res. 2016; 69: 2318–2324. Publisher Full Text\n\nThomée S, Härenstam A, Hagberg M: Mobile phone use and stress, sleep disturbances, and symptoms of depression among young adults - a prospective cohort study. BMC Public Health. 2011; 11: 66. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGunawan J, Juthamanee S, Aungsuroch Y: Current Mental Health Issues in the Era of Covid-19. Asian J Psychiatr. 2020; 51: 102103. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTorales J, O’Higgins M, Castaldelli-Maia JM, et al.: The outbreak of COVID-19 coronavirus and its impact on global mental health. Int J Soc Psychiatry. 2020; PubMed Abstract \| Publisher Full Text\n\nArdito L, Scuotto V, Del Giudice M, et al.: A bibliometric analysis of research on Big Data analytics for business and management. Manag Decis 2019; 57: 1993–2009. Publisher Full Text\n\nBornmann L: “Bibliometric Indicators,” In: SAGE Research Methods Foundations , eds. Atkinson P, Delamont S, Cernat A, et al.: 1 Oliver’s Yard, 55 City Road, London EC1Y 1SP United Kingdom: SAGE Publications Ltd. Publisher Full Text\n\nLöhönen J, Isohanni M, Nieminen P, et al.: Coverage of the bibliographic databases in mental health research. Nord J Psychiatry. 2010; 64: 181–188. PubMed Abstract \| Publisher Full Text\n\nNatsiavas P, Maglaveras N, Koutkias V: A public health surveillance platform exploiting free-text sources via natural language processing and linked data: Application in adverse drug reaction signal detection using PubMed and Twitter. In: Lecture Notes in Computer Science (including subseries Lecture Notes in Artificial Intelligence and Lecture Notes in Bioinformatics) . Publisher Full Text\n\nBurnham JF: Scopus database: a review. Biomed Digit Libr. 2006; 3(1). Publisher Full Text\n\nMontoya FG, Alcayde A, Baños R, et al.: A fast method for identifying worldwide scientific collaborations using the Scopus database. Telemat Informatics. 2018; 35: 168–185. Publisher Full Text\n\nJames C, Colledge L, Meester W, et al.: CiteScore metrics: Creating journal metrics from the Scopus citation index. Learn Publ. 2019; 32: 367–374. Publisher Full Text\n\nRoldan-Valadez E, Salazar-Ruiz SY, Ibarra-Contreras R, et al.: Current concepts on bibliometrics: a brief review about impact factor, Eigenfactor score, CiteScore, SCImago Journal Rank, Source-Normalised Impact per Paper, H-index, and alternative metrics. Irish J Med Sci (1971). 2019; 188: 939–951. PubMed Abstract \| Publisher Full Text\n\nSchou L, Høstrup H, Lyngsø EE, et al.: Validation of a new assessment tool for qualitative research articles. J Adv Nurs. 2012; 68: 2086–2094. PubMed Abstract \| Publisher Full Text\n\nKim YJ, Qian L, Aslam MS: Development of a Personalized Mobile Mental Health Intervention for Workplace Cyberbullying Among Health Practitioners: Protocol for a Mixed Methods Study. JMIR Res Protoc. 2020; 9: e23112.PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilliams AE: F1000: an overview and evaluation. Inf Learn Sci. 2017; 118: 364–371. Publisher Full Text\n\nSicilia M-A, García-Barriocanal E, Sánchez-Alonso S: Community Curation in Open Dataset Repositories: Insights from Zenodo. Procedia Comput Sci. 2017; 106: 54–60. Publisher Full Text\n\nO’Brien K: ResearchGate. J Med Libr Assoc. 2019: 107. Publisher Full Text" }	[ { "id": "85390", "date": "03 Jun 2021", "name": "Peter Kokol", "expertise": [ "Reviewer Expertise Bibliometrics", "knowledge synthesis", "computer science" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors didn't provide information on how the corpora from Pubmed and Scopus will be integrated, actually, PubMed is mentioned only in the introduction and not in the data extraction.\n\nThe presented search strings will probably result in empty corpora, due to too many ANDs in the strings.\n\nThe same search strings work differently in Sopus than in Pubmed.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? No\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [] }, { "id": "94289", "date": "09 Nov 2021", "name": "Ludo Waltman", "expertise": [ "Reviewer Expertise Bibliometrics", "scientometrics", "research evaluation" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nWhile I appreciate the efforts made by the authors to publish a protocol of their study, I am afraid I am not convinced that publishing a protocol is very useful for this specific type of study. Bibliometric analyses like the one proposed by the authors are of an exploratory (rather than confirmatory) nature. I, therefore, question the usefulness of publishing a study protocol. A bibliometric analysis like this one will inevitably require a number of iterations in which the search queries and other aspects of the methodological design are gradually being improved. It is for instance not possible to assess the quality of the search queries without observing the results obtained from the queries. I, therefore, feel that I am not able to evaluate this study protocol. For research projects that are of a confirmatory nature, publishing a study protocol can be a great way to improve the quality and reliability of the research, but for projects that are of a more exploratory nature, like this one, I do not see a clear benefit in publishing a study protocol and I am not sure how I can evaluate such a protocol.\nI have decided not to approve this protocol. I would like to emphasize that this decision does not reflect any specific concerns I have about the content of the protocol under review. Instead, it reflects my general discomfort about evaluating a protocol for studies that are of an exploratory nature.\n\nIs the rationale for, and objectives of, the study clearly described? No\n\nIs the study design appropriate for the research question? No\n\nAre sufficient details of the methods provided to allow replication by others? No\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [] } ]	1	https://f1000research.com/articles/10-225

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